Method for inhibiting or disrupting biofilm formation, or reducing biofilm

ABSTRACT

Cationic pillar[n]arenes, e.g., positively charged poly-ammonium, poly-phosphonium and poly-imidazolium pillar[5-6]arene derivatives are capable of inhibiting or preventing biofilm formation, and facilitating existing biofilm decomposition. A composition for inhibiting or disrupting biofilm, e.g., bacterial or fungal biofilm, formation, or reducing biofilm, can include a pharmaceutically acceptable carrier, and a cationic pillar[n]ene.

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

Any and all applications for which a foreign or domestic priority claimis identified in the Application Data Sheet as filed with the presentapplication are hereby incorporated by reference under 37 CFR 1.57.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to cationic pillar[n]arenes, e.g.,cationic pillar[5-6]arenes, and uses thereof in inhibiting or disruptingbiofilm formation, or reducing biofilm.

Description of the Related Art

Bacterial biofilms are defined as microbial communities (cities ofmicrobes) that are held together by an extracellular matrix. In recentyears, there is an increasing interest in bacterial biofilms as a resultof the fact that in the majority of the cases biofilms lead to adramatic enhancement in resistance to antimicrobial agents (Fux et al.,2005; Davies, 2003; Rabin et al., 2015). Compared with planktonicbacteria (bacteria that grow in suspension), biofilm forming bacteriacan be up to almost three orders of magnitude less susceptible toantibiotics (Bottcher et al., 2013). Moreover, it was estimated thatbiofilms account for a large percentage of nosocomial and implanteddevice-derived microbial infections in patients (Davey and O'Toole,2000).

Biofilm matrices are composed of exo-polymeric substances (EPS) that arehigh-molecular weight compounds secreted by the bacteria into theextracellular environment and are crucial for the integrity of allbiofilms (Bottcher et al., 2013; Davey and O'Toole, 2000). EPScomponents include polysaccharides also termed exo-polysaccharides,proteins, extracellular DNA, lipids and bacterial decompositionsubstances that are held together by a highly complex network ofhydrogen bonds as well as ionic and van der Waals interactions betweenthe different matrix components. The composition of biofilm matricesvaries significantly amongst different bacterial strains; some matricescontain mainly exo-polysaccharides and some mainly proteins. Moreover,the structure of the proteins and the monosaccharide building blocksthat compose exo-polysaccharides vary between different biofilmproducing bacterial strains.

Investigation of the biofilm formation process in the Gram negativePseudomonas aeruginosa which is one of the major causes for lethalbacterial lung infections revealed a biofilm formation process that wasgenerally defined as a five step sequence described in FIG. 1 (Jenningset al., 2014). In the first stage, planktonic bacteria adhere to thesurface on which the biofilm is about to form. At this point, most ofthe interactions with the surface are based on van-der Waals reversibleforces and the bacterial cell can easily leave the surface back into themedia. Once these weak initial interactions took place, in the secondstage, the bacterial cells more permanently anchor themselves to thesurface by a cell adhesion process that involves protein basedinteractions. In the third and fourth stages, additional bacterial celllayers adhere to the first layer and form micro colonies that continueto grow and mature by forming an extracellular matrix coating. In thefinal fifth stage, biofilm forming bacterial cells leave the maturebiofilm into the environment and remain as planktonic bacteria orestablish new biofilm colonies depending on the environmentalconditions.

Clardy and coworkers previously reported a collection of syntheticguanidine- and bi-guanidine-based cationic amphiphiles that inhibitedbiofilm formation as well as eradicated existing biofilms of Bacillussubtilis and Staphylococcus aureus strains (Bottcher et al., 2013). Morerecently, Wuest and coworkers reported a collection of quaternaryammonium amphiphiles (Jennings et al., 2014) that demonstratedantimicrobial activity against a collection of Gram positive and Gramnegative bacterial strains. In addition, some of these cationicamphiphiles efficiently broke down existing biofilms of the two Grampositive pathogens Staphylococcus aureus and Enterococcus faecalis.However it was reported that these molecules are all hemolytic compoundsexhibiting pronounced toxicity against mammalian cells (Jennings et al.,2014).

Pillar[n]arenes, first reported in 2008 (Ogoshi et al., 2008), havesymmetrical cylindrical structures and relatively large free volumes.Pillar[n]arenes can be obtained and functionalized by simple and highyield synthesis routes making them a versatile macrocycles for variousapplications. These macrocycles possess host-guest properties owing totheir π-electron rich cavity and crown ether-like arrangement of oxygenatoms at both rims. Hence, in recent years, pillar[n]arenes have beenused in host-guest chemistry and as sensors and were used to constructsupramolecular polymers, interlocked molecules, and hybrid biomolecularmaterials (Ogoshi et al., 2008; Ogoshi and Yamagishi, 2014; Cragg andSharma, 2012; Xue et al., 2012; Dong et al., 2014a; Chunju, 2014; Ogoshiet al., 2016; Ma et al., 2016; Liz et al., 2016; Shi et al., 2016;Ogoshi et al., 2015; Nierengarten et al., 2013; Li, 2014; Yang et al.,2014; Adiri et al., 2013; Zhang and Zhao, 2013; Yao et al., 2014; Donget al., 2014b; Mao et al., 2016; Wang et al., 2015; Jie et al., 2014).Despite the significant attention that pillararenes have received fromthe chemical community, to date, their biological activity remainsrelatively unexplored.

Despite the great need, there are currently no clinically approved smallmolecules that act as efficient inhibitors of biofilm formation and/oras eradicators of mature biofilms, without affecting bacterial cellviability. Identification of such small molecules will offer a muchneeded solution to biofilm infections while not affecting the importantnatural bacterial flora of the body. In addition, it is likely that thebacteria will not develop any defense mechanisms against such biofilminhibitors or eradicators.

SUMMARY OF INVENTION

In one aspect, the present invention provides a compound of the formulaI:

-   -   wherein    -   R₁ is —CR₆R₇—, wherein R₆ and R₇ each independently is H,        halogen, —COR₈, —COOR₈, —OCOOR₈, —OCON(R₈)₂, —CN, —NO₂, —SR₈,        —OR₈, —N(R₈)₂, —CON(R₈)₂, —SO₂R₈, —SO₃H, —S(═O)R₈, or        (C₁-C₈)alkyl optionally substituted by one or more groups each        independently selected from —COR₈, —COOR₈, —OCOOR₈, —OCON(R₈)₂,        —CN, —NO₂, —SR₈, —OR₈, —N(R₈)₂, —CON(R₈)₂, —SO₂R₈, —SO₃H,        —S(═O)R₈, —N⁺(R′)₃ or —P⁺(R′)₃, wherein R′ each independently is        H, (C₁-C₆)alkyl, phenyl, benzyl, or heterocyclyl, or two R's        together with the N atom to which they are attached form a 3-7        membered saturated ring, optionally containing one or more        heteroatoms selected from O, S or N and optionally further        substituted at the additional N atom;    -   R₂ and R₃ each independently is H, halogen, or (C₁-C₈)alkyl        optionally substituted by one or more groups each independently        selected from halogen, —COR₈, —COOR₈, —OCOOR₈, —OCON(R₈)₂, —CN,        —NO₂, —SR₈, —OR₈, —N(R₈)₂, —CON(R₈)₂, —SO₂R₈, —SO₃H, —S(═O)R₈,        —N⁺(R′)₃ or —P⁺(R′)₃, wherein R′ each independently is H,        (C₁-C₆)alkyl, phenyl, benzyl, or heterocyclyl, or two R's        together with the N atom to which they are attached form a 3-7        membered saturated ring, optionally containing one or more        heteroatoms selected from O, S or N and optionally further        substituted at the additional N atom;    -   R₄ and R₅ each independently is selected from (C₁-C₁₀)alkylene,        (C₂-C₁₀)alkenylene, or (C₂-C₁₀)alkynylene, optionally        substituted by one or more groups each independently selected        from halogen, —COR₈, —COOR₈, —OCOOR₈, —OCON(R₈)₂, —CN, —NO₂,        —SR₈, —OR₈, —N(R₈)₂, —CON(R₈)₂, —SO₂R₈, —SO₃H, —S(═O)R₈,        (C₆-C₁₀)aryl, (C₁-C₄)alkylene-(C₆-C₁₀)aryl, heteroaryl, or        (C₁-C₄)alkylene-heteroaryl, and further optionally interrupted        by one or more identical or different heteroatoms selected from        S, O or N, and/or at least one group each independently selected        from —NH—CO—, —CO—NH—, —N(C₁-C₈alkyl)-, —N(C₆-C₁₀aryl)-,        (C₆-C₁₀)arylenediyl, or heteroarylenediyl;    -   R₈ each independently is H or (C₁-C₈)alkyl;    -   Y each independently is a cation derived from a        nitrogen-containing group, a nitrogen-containing mono- or        polycyclic heteroaromatic group optionally containing O, S or        additional N atoms, or an onium group not containing nitrogen,        linked to R₄ or R₅ via its positively charged atom;    -   X is a counter anion such as Br⁻, Cl⁻, F⁻, I⁻, PF₆ ⁻, BF₄ ⁻,        OH⁻, ClO₄ ⁻, HSO₄ ⁻, CF₃COO⁻, CN⁻, alkylCOO⁻, arylCOO⁻, a        pharmaceutically acceptable anion, or a combination thereof; and    -   n is an integer of 5-11,    -   but excluding the compounds wherein R₁ is —CH₂—; R₂ and R₃ are        H; and: (i) n is 5; R₄ and R₅ are —(CH₂)₂—; and Y is        1-methyl-imidazolium-3-yl or —N⁺(CH₃)₃; (ii) n is 5; R₄ and R₅        are —(CH₂)₃—; and Y is —P⁺(C₄H₉)₃; (iii) n is 5; R₄ and R₅ are        —(CH₂)₄—; and Y is —N⁺(CH₃)₃; (iv) n is 6; R₄ and R₅ are        —(CH₂)₂—; and Y is —N⁺(CH₃)₃; (v) n is 6; R₄ and R₅ are        —(CH₂)₄—; and Y is —N⁺(CH₃)₃; (vi) n is 6; R₄ and R₅ are        —(CH₂)₄—; and Y is 1-pyridinium; or (vii) n is 6; R₄ and R₅ are        —(CH₂)₂—; and Y is 1-methyl-imidazolium-3-yl.

More particularly, the invention provides a compound of the formula I asdefined above, wherein Y each independently is (i) a cation derived froma nitrogen-containing group and selected from an ammonium [—N⁺(R′)₃],hydrazinium [—N⁺(R′)₂—N(R′)₂], ammoniumoxy [—N⁺(R′)₂—O], iminium[—N⁺(R′)₂═C<], amidinium [—N⁺(R′)₂—C(R′)═NR′], or guanidinium[—N⁺(R′)₂—C(═NR′)—N(R′)₂]; (ii) a cation derived from anitrogen-containing mono- or polycyclic heteroaromatic group andselected from pyrazolium, imidazolium, oxazolium, thiazolium,pyridinium, pyrimidinium, quinolinium, isoquinolinium, 1,2,4-triazinium,1,3,5-triazinium, or purinium, optionally substituted by one or moregroups each independently selected from halogen, (C₁-C₆)alkyl, —COH,—COOH, —OCOOH, —OCONH₂, —CN, —NO₂, —SH, —OH, —NH₂, —CONH₂, —SO₃H, —SO₂H,or —S(═O)H; or (iii) a cation derived from an onium group not containingnitrogen and selected from phosphonium [—P⁺(R′)₃], arsonium [—As⁺(R′)₃],oxonium [—O⁺(R′)₂], sulfonium [—S⁺(R′)₂], selenonium [—Se⁺(R′)₂],telluronium [—Te⁺(R′)₂], stibonium [—Sb⁺(R′)₃], or bismuthonium[—Bi⁺(R′)₃], wherein R′ each independently is H, (C₁-C₆)alkyl, phenyl,benzyl, or heterocyclyl, or two R's in the ammonium, hydrazinium,ammoniumoxy, iminium, amidinium or guanidinium groups, together with theN atom to which they are attached, form a 3-7 membered saturated ring,optionally containing one or more heteroatoms selected from 0, S or Nand optionally further substituted at the additional N atom.

In another aspect, the present invention provides a compositioncomprising a compound of the formula I as defined above, i.e., excludingthe compounds excluded by the proviso above, and a carrier.

In a further aspect, the present invention relates to a method forinhibiting or disrupting biofilm formation in an aqueous media or on anobject, or for reducing biofilm existing in an aqueous media or attachedto an object, said method comprising contacting said aqueous media orobject with a compound of the formula I, including the compoundsexcluded by the proviso above.

In certain embodiments, the method of the present invention is forinhibiting or disrupting biofilm formation in an aqueous media, or forreducing biofilm existing in said aqueous media, and comprises in factdissolving of said compound or a composition comprising it within saidaqueous media. In other embodiments, the method of the present inventionis for inhibiting or disrupting biofilm formation on an object, or forreducing biofilm attached to said object, and comprises coating of saidobject with said compound or a composition comprising it, or immersingof said object within a composition comprising said compound,respectively.

In yet another aspect, the present invention relates to a pharmaceuticalcomposition for inhibiting or disrupting biofilm, e.g., bacterial orfungal biofilm, formation, or reducing biofilm, said compositioncomprising a pharmaceutically acceptable carrier, and a compound of theformula I wherein X is a pharmaceutically acceptable anion, includingthe compounds excluded by the proviso above.

In still another aspect, the present invention relates to a compound ofthe formula I, including the compounds excluded by the proviso above,for use in inhibiting or disrupting biofilm, e.g., bacterial or fungalbiofilm, formation, or reducing biofilm.

In yet a further aspect, the present invention relates to a method forinhibiting or disrupting biofilm formation, or reducing biofilm, in anindividual in need thereof, comprising administering to said individuala therapeutically effective amount of a compound of the formula I,including the compounds excluded by the proviso above.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates the stages of biofilm formation process.

FIG. 2 shows inhibition of biofilm formation by pillar[6]arene 27.Biofilms produced by S. aureus subsp. aureus Rosenbach ATCC 33592 (a)and by E. faecalis ATCC 29212 (b) in the presence of increasingconcentrations of 27 were stained with crystal violet. Eachconcentration of compound was tested in five wells.

FIGS. 3A-3B show biofilm formation by E. faecalis ATCC 29212 (3A) and S.aureus subsp. aureus Rosenbach ATCC 33592 (3B), evaluated using thedouble-dilution method with starter inoculum of 1:100 (OD₆₀₀=0.01).Molarity concentration ranges of the tested compounds: (21) 0.22-28.18;(22) 0.20-25.59; (23) 0.18-23.78; (25) 0.21-26.54; (26) 0.27-35.00; and(27) 0.18-23.48 μM. No measurable biofilm inhibition effect was detectedfor compounds 24 and 5 up to concentrations of 24.88 and 45.36 μM,respectively.

FIGS. 4A-4C show the effect of initial inoculum on biofilm formationinhibition by compound 27. The compound was incubated with E. faecalis(ATCC 29212; right panels) and S. aureus subsp. aureus Rosenbach (ATCC33592; left panels) at starter inoculums of OD₆₀₀ 0.025 (4A), 0.05 (4B),or 0.10 (4C).

FIGS. 5A-5D show growth curves of bacteria in the presence of compound27: (5A) E. coli ATCC 25922, (5B) P. aeruginosa PAO1, (5C) E. faecalisATCC 29212, and (5D) S. aureus subsp. aureus Rosenbach ATCC 33592.Bacteria were incubated with compound 27 (32 and 64 μg/mL) for 24 h at37° C. Pillar[6]arene conjugate 27 did not inhibit the growth of thesebacteria.

FIGS. 6A-6B show the effect of compound 27 on the viability of IB3-1cells (6A); and THP-1 cells (6B).

FIG. 7 shows biofilms of S. aureus subsp. aureus Rosenbach ATCC 33592 inthe presence of increasing concentrations (μM) of pillar[5]arenederivatives (a) compound 29, (b) compound 30, (c) compound 25, and (d)compound 28 stained with crystal violet.

FIG. 8 shows biofilms formed by E. faecalis ATCC 29212 in the presenceof increasing concentrations (μM) of pillar[5]arene derivatives (a)compound 29, (b) compound 30, (c) compound 25, and (d) compound 28stained with crystal violet.

FIG. 9 shows biofilms formed by S. aureus subsp. aureus Rosenbach ATCC33592 in the presence of increasing concentrations of (a) compound 12and (c) compound 11. Biofilms formed by E. faecalis ATCC 29212 in thepresence of increasing concentrations of (b) compound 12 and (d)compound 11. All the wells were stained with crystal violet.

FIGS. 10A-10C show biofilm formation by S. aureus ATCC 33592 (MRSA)(10A) and E. faecalis ATCC 29212 (10B) evaluated using thedouble-dilution method with starter inoculum of 1:100 (OD600=0.01) inthe presence of compounds 25 and 28-30; and biofilm formation in thepresence of compounds 11 and 12 (10C) Concentration ranges of the testedcompounds were: (25) 0.21-13.27; (28) 0.18-11.30; (29) 0.19-12.40; (30)0.17-10.66; (11) 5.3-340 μM; and (12) 4.95-317.

FIGS. 11A-11B show i-H NMR spectra of compound 28 after incubation pH(a) 7.4, (b) 2.3, and (c) 10.2 for 4 hours (11A); and ¹H NMR spectra ofcompound 30 after incubation at pH (a) 7.4, (b) 2.3, and (c) 10.7 for 4hours (11B).

FIGS. 12A-12B show biofilm formation by S. aureus subsp. aureusRosenbach ATCC 33592 (12A) and E. faecalis ATCC 29212 (12B) in thepresence of increasing concentrations (μM) 28 and 30 that had beenincubated for 4 hours at different pH levels.

FIGS. 13A-13B show the cell toxicity of compounds 25, 29 and 30 onmammalian IB3-1 cell-line (13A) and HaCaT cell-line (13B).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention relates to cationic amphiphilic systems capable ofinterfering with the chemical interactions between different componentsof biofilm matrices, more particularly bacterial biofilm matrices,thereby inhibiting or preventing biofilm formation and facilitatingexisting biofilm decomposition without intervening with bacterialviability and damaging mammalian cell membranes as many families ofcationic amphiphiles do.

More specifically, the invention provides cationic pillararenederivatives such as positively charged poly-ammonium, poly-phosphoniumand poly-imidazolium pillararene derivatives. These systems are watersoluble organic salts containing both a lipophilic but relativelyelectron-rich cavity as well as two arrays of positively chargedmoieties of the two opposite faces of the pillararene backbone which caninteract with the negatively charged membranes and other differentmolecular components of the biofilm matrices. The cationicammonium/phosphonium pillar[5-6]arenes exemplified herein, includingboth known and novel compounds, were synthesized according to theprocedures described in detail in the Experimental Section and depictedin Schemes 1-3 hereinafter, and are shown in Scheme 4.

The novel cationic pillar[5-6]arene derivatives specifically disclosedherein are compounds of the formula I, wherein (i) n is 5; R₁ is —CH₂—;R₂ and R₃ are H; R₄ and R₅ are —(CH₂)₃—; and Y is —N⁺(CH₃)₃ or—P⁺(CH₃)₃, herein identified compound 25 and 29, respectively; (ii) n is5; R₁ is —CH₂—; R₂ and R₃ are H; R₄ and R₅ are —(CH₂)₃—; and Y is—N⁺(C₂H₅)₃ or —P⁺(C₂H₅)₃, herein identified compound 28 and 30,respectively; (iii) n is 5; R₁ is —CH₂—; R₂ and R₃ are H; R₄ and R₅ are—(CH₂)₆—, and Y is —N⁺(CH₃)₃, herein identified compound 31; or (iv) nis 6; R₁ is —CH₂—; R₂ and R₃ are H; R₄ and R₅ are —(CH₂)₃—, and Y is—N⁺(CH₃)₃, herein identified compound 32. Additional cationicpillar[5-6]arene derivatives specifically exemplified are compounds ofthe formula I, wherein (i) n is 5; R₁ is —CH₂—; R₂ and R₃ are H; R₄ andR₅ are —(CH₂)₂—; and Y is —N⁺(CH₃)₃, —N⁺(C₂H₅)₃, or1-methyl-imidazolium-3-yl, herein identified compound 21 (or 26), 23 and22, respectively; or (ii) n is 6; R₁ is —CH₂—; R₂ and R₃ are H; R₄ andR₅ are —(CH₂)₂—, and Y is —N⁺(CH₃)₃, or 1-methyl-imidazolium-3-yl,herein identified compound 27 and 33, respectively. In all thosecompounds, X is Br⁻ or Cl⁻.

As has been found and shown herein, the water soluble cationicpillar[5-6]arene derivatives tested are extremely efficient ininhibiting biofilm formation at sub μM concentrations, without affectingthe tested bacterial cell viability or causing measurable damage to themembranes of mammalian red blood cells (RBCs). The phosphonium-decoratedpillararenes exhibited similar potencies as inhibitors of biofilmformation as their corresponding ammonium analogues, demonstrating thatthe number of positively charged groups and not their chemical identityare key to their anti-biofilm activity. The pillararene platform appearsto be important and positive charges operating cooperatively are neededfor effective anti-biofilm activity, as indicated by our finding thatthe respective cationic monomers were completely inactive.Interestingly, the cationic pillararene derivatives tested retainedtheir anti-biofilm capability even after four hours of exposure toacidic or alkaline pH. The organisms in which the compounds were foundto inhibit biofilm formation include clinical pathogens such asMethicillin-resistant Staphylococcus aureus (MRSA), Pseudomonasaeruginosa and more.

Such cationic pillararene derivatives can thus be used to fight biofilmformation and to eradicate existing biofilms in myriad of applicationssuch as in water reservoirs, closed circuit water systems, in paintingindustries especially in dyes used to protect vessels from water, intoothpaste and dentistry industry, and more. The compounds may also beused to fight biofilm on surfaces in hospitals, medical devices andimplants, as well as in external and internal pads and optionally inconjunction with antibiotic treatments.

In one aspect, the present invention thus provides a compound of theformula I:

-   -   wherein    -   R₁ is —CR₆R₇—, wherein R₆ and R₇ each independently is H,        halogen, —COR₈, —COOR₈, —OCOOR₈, —OCON(R₈)₂, —CN, —NO₂, —SR₈,        —OR₈, —N(R₈)₂, —CON(R₈)₂, —SO₂R₈, —SO₃H, —S(═O)R₈, or        (C₁-C₈)alkyl optionally substituted by one or more groups each        independently selected from —COR₈, —COOR₈, —OCOOR₈, —OCON(R₈)₂,        —CN, —NO₂, —SR₈, —OR₈, —N(R₈)₂, —CON(R₈)₂, —SO₂R₈, —SO₃H,        —S(═O)R₈, —N⁺(R′)₃ or —P⁺(R′)₃, wherein R′ each independently is        H, (C₁-C₆)alkyl, phenyl, benzyl, or heterocyclyl, or two R's        together with the N atom to which they are attached form a 3-7        membered saturated ring, optionally containing one or more        heteroatoms selected from O, S or N and optionally further        substituted at the additional N atom;    -   R₂ and R₃ each independently is H, halogen, or (C₁-C₈)alkyl        optionally substituted by one or more groups each independently        selected from halogen, —COR₈, —COOR₈, —OCOOR₈, —OCON(R₈)₂, —CN,        —NO₂, —SR₈, —OR₈, —N(R₈)₂, —CON(R₈)₂, —SO₂R₈, —SO₃H, —S(═O)R₈,        —N⁺(R′)₃ or —P⁺(R′)₃, wherein R′ each independently is H,        (C₁-C₆)alkyl, phenyl, benzyl, or heterocyclyl, or two R's        together with the N atom to which they are attached form a 3-7        membered saturated ring, optionally containing one or more        heteroatoms selected from O, S or N and optionally further        substituted at the additional N atom;    -   R₄ and R₅ each independently is selected from (C₁-C₁₀)alkylene,        (C₂-C₁₀)alkenylene, or (C₂-C₁₀)alkynylene, optionally        substituted by one or more groups each independently selected        from halogen, —COR₈, —COOR₈, —OCOOR₈, —OCON(R₈)₂, —CN, —NO₂,        —SR₈, —OR₈, —N(R₈)₂, —CON(R₈)₂, —SO₂R₈, —SO₃H, —S(═O)R₈,        (C₆-C₁₀)aryl, (C₁-C₄)alkylene-(C₆-C₁₀)aryl, heteroaryl, or        (C₁-C₄)alkylene-heteroaryl, and further optionally interrupted        by one or more identical or different heteroatoms selected from        S, O or N, and/or at least one group each independently selected        from —NH—CO—, —CO—NH—, —N(C₁-C₈alkyl)-, —N(C₆-C₁₀aryl)-,        (C₆-C₁₀)arylenediyl, or heteroarylenediyl;    -   R₈ each independently is H or (C₁-C₈)alkyl;    -   Y each independently is (i) a cation derived from a        nitrogen-containing group and selected from an ammonium        [—N⁺(R′)₃], hydrazinium [—N⁺(R′)₂—N(R′)₂], ammoniumoxy        [—N⁺(R′)₂—O], iminium [—N⁺(R′)₂═C<], amidinium        [—N⁺(R′)₂—C(R′)═NR′], or guanidinium        [—N⁺(R′)₂—C(═NR′)—N(R′)₂]; (ii) a cation derived from a        nitrogen-containing mono- or polycyclic heteroaromatic group and        selected from pyrazolium, imidazolium, oxazolium, thiazolium,        pyridinium, pyrimidinium, quinolinium, isoquinolinium,        1,2,4-triazinium, 1,3,5-triazinium, or purinium, optionally        substituted by one or more groups each independently selected        from halogen, (C₁-C₆)alkyl, —COH, —COOH, —OCOOH, —OCONH₂, —CN,        —NO₂, —SH, —OH, —NH₂, —CONH₂, —SO₃H, —SO₂H, or —S(═O)H; or (iii)        a cation derived from an onium group not containing nitrogen and        selected from phosphonium [—P⁺(R′)₃], arsonium [—As⁺(R′)₃],        oxonium [—O⁺(R′)₂], sulfonium [—S⁺(R′)₂], selenonium        [—Se⁺(R′)₂], telluronium [—Te⁺(R′)₂], stibonium [—Sb⁺(R′)₃], or        bismuthonium [—Bi⁺(R′)₃], wherein R′ each independently is H,        (C₁-C₆)alkyl, phenyl, benzyl, or heterocyclyl, or two R's in the        ammonium, hydrazinium, ammoniumoxy, iminium, amidinium or        guanidinium groups, together with the N atom to which they are        attached, form a 3-7 membered saturated ring, optionally        containing one or more heteroatoms selected from O, S or N and        optionally further substituted at the additional N atom;    -   X is a counter anion such as Br⁻, Cl⁻, F⁻, I⁻, PF₆ ⁻, BF₄ ⁻,        OH⁻, ClO₄ ⁻, HSO₄ ⁻, CF₃COO⁻, CN⁻, alkylCOO⁻, arylCOO⁻, a        pharmaceutically acceptable anion, or a combination thereof; and    -   n is an integer of 5-11,    -   but excluding the compounds wherein R₁ is —CH₂—; R₂ and R₃ are        H; and: (i) n is 5; R₄ and R₅ are —(CH₂)₂—; and Y is        1-methyl-imidazolium-3-yl or —N⁺(CH₃)₃; (ii) n is 5; R₄ and R₅        are —(CH₂)₃—; and Y is —P⁺(C₄H₉)₃; (iii) n is 5; R₄ and R₅ are        —(CH₂)₄—; and Y is —N⁺(CH₃)₃; (iv) n is 6; R₄ and R₅ are        —(CH₂)₂—; and Y is —N⁺(CH₃)₃; (v) n is 6; R₄ and R₅ are        —(CH₂)₄—; and Y is —N⁺(CH₃)₃; (vi) n is 6; R₄ and R₅ are        —(CH₂)₄—; and Y is 1-pyridinium; or (vii) n is 6; R₄ and R₅ are        —(CH₂)₂—; and Y is 1-methyl-imidazolium-3-yl.

The term “halogen” as used herein refers to a halogen and includesfluoro, chloro, bromo, and iodo, and it is preferably chloro or bromo.

The term “alkyl” as used herein typically means a linear or branchedsaturated hydrocarbon radical having 1-8 carbon atoms and includes,e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,tert-butyl, n-pentyl, isoamyl, 2,2-dimethylpropyl, n-hexyl, n-heptyl,n-octyl, and the like. Preferred are (C₁-C₆)alkyl groups, morepreferably (C₁-C₄)alkyl groups, most preferably methyl, ethyl or propyl.The alkyl defined herein may optionally be substituted with one or moregroups each independently selected from halogen, —COR, —COOR, —OCOOR,—OCON(R)₂, —CN, —NO₂, —SR, —OR, —N(R)₂, —CON(R)₂, —SO₂R, —SO₃R or—S(═O)R, wherein R is H or unsubstituted (C₁-C₈)alkyl.

The term “alkylene” as used herein typically means a straight orbranched divalent hydrocarbon radical having 1-10 carbon atoms, e.g.,methylene, ethylene, propylene, butylene, 2-methylpropylene, pentylene,2-methylbutylene, hexylene, 2-methylpentylene, 3-methylpentylene,2,3-dimethylbutylene, heptylene, octylene, nonylene, decanylene, and thelike. Preferred are (C₁-C₈)alkylene or (C₁-C₆)alkylene, more preferably(C₁-C₄)alkylene, most preferably methylene, ethylene, propylene, orbutylene. The terms “alkenylene” and “alkynylene” typically meanstraight or branched divalent hydrocarbon radicals having 2-10 carbonatoms, and one or more double or triple bonds, respectively.

The term “aryl” denotes an aromatic carbocyclic group having 6-10 carbonatoms consisting of a single ring or condensed multiple rings such as,but not limited to, phenyl and naphthyl. The aryl defined herein mayoptionally be substituted with one or more groups each independentlyselected from halogen, —COR, —COOR, —OCOOR, —OCON(R)₂, —CN, —NO₂, —SR,—OR, —N(R)₂, —CON(R)₂, —SO₂R, —SO₃R, —S(═O)R, or —(C₁-C₈)alkyl, whereinR is H or unsubstituted (C₁-C₈)alkyl. The term “arylenediyl” refers to adivalent radical derived from an “aryl” as defined herein by removal ofa further hydrogen atom from any of the ring atoms.

The term “heterocyclic ring” denotes a mono- or poly-cyclic non-aromaticring of 4-12 atoms containing at least one carbon atom and one to threeheteroatoms selected from sulfur, oxygen or nitrogen, which may besaturated or unsaturated, i.e., containing at least one unsaturatedbond. Preferred are 3- or 7-membered heterocyclic rings. The term“heterocyclyl” as used herein refers to any univalent radical derivedfrom a heterocyclic ring as defined herein by removal of hydrogen fromany ring atom. Examples of such radicals include, without limitation,piperidino, 4-morpholinyl, or pyrrolidinyl. The heterocyclyl definedherein may optionally be substituted, at any position of the ring, withone or more groups each independently selected from halogen, —COR,—COOR, —OCOOR, —OCON(R)₂, —CN, —NO₂, —SR, —OR, —N(R)₂, —CON(R)₂, —SO₂R,—SO₃R, —S(═O)R, or —(C₁-C₈)alkyl, wherein R is H or unsubstituted(C₁-C₈)alkyl.

The term “heteroaryl” refers to a radical derived from a 5-10-memberedmono- or poly-cyclic heteroaromatic ring containing 1-3, preferably 1-2,heteroatoms selected from nitrogen, sulfur or oxygen. Examples ofmono-cyclic heteroaryls include, without being limited to, pyrrolyl,furyl, thienyl, thiazinyl, pyrazolyl, pyrazinyl, imidazolyl, oxazolyl,isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl,1,2,3-triazinyl, 1,3,4-triazinyl, and 1,3,5-triazinyl. Polycyclicheteroaryl radicals are preferably composed of two rings such as, butnot limited to, benzofuryl, isobenzofuryl, benzothienyl, indolyl,quinolinyl, isoquinolinyl, imidazo[1,2-a]pyridyl, benzimidazolyl,benzthiazolyl, benzoxazolyl, pyrido[1,2-a]pyrimidinyl and1,3-benzodioxinyl. The heteroaryl may optionally be substituted by oneor more groups each independently selected from halogen, —COR, —COOR,—OCOOR, —OCON(R)₂, —CN, —NO₂, —SR, —OR, —N(R)₂, —CON(R)₂, —SO₂R, —SO₃R,—S(═O)R, or —(C₁-C₈)alkyl, wherein R is H or unsubstituted (C₁-C₈)alkyl.It is to be understood that when a polycyclic heteroaryl is substituted,the substitution may be in any of the carbocyclic and/or heterocyclicrings. The term “heteroarylenediyl” denotes a divalent radical derivedfrom a “heteroaryl” as defined herein by removal of a further hydrogenatom from any of the ring atoms.

The term “cation derived from a nitrogen-containing group” as usedherein denotes for example, but without limiting to, an ammonium[—N⁺(R′)₃], hydrazinium [—N⁺(R′)₂—N(R′)₂], ammoniumoxy [—N⁺(R′)₂→O],iminium [—N⁺(R′)₂═C<], amidinium [—N⁺(R′)₂—C(R′)═NR′], or guanidinium[—N⁺(R′)₂—C(═NR′)—N(R′)₂], wherein R′ each independently is H,(C₁-C₆)alkyl, phenyl, benzyl, or heterocyclyl, or two R's together withthe N atom to which they are attached form a 3-7 membered saturatedring, optionally containing one or more heteroatoms selected from 0, Sor N and optionally further substituted at the additional N atom. Inparticular embodiments, the cation derived from a nitrogen-containinggroup is an ammonium as defined hereinabove.

The term “cation derived from a nitrogen-containing mono- or polycyclicheteroaromatic group optionally containing 0, S or additional N atoms”as used herein denotes for example, but without limiting to, pyrazolium,imidazolium, oxazolium, thiazolium, pyridinium, pyrimidinium,quinolinium, isoquinolinium, 1,2,4-triazinium, 1,3,5-triazinium, orpurinium, optionally substituted by one or more groups eachindependently selected from halogen, (C₁-C₆)alkyl, —COH, —COOH, —OCOOH,—OCONH₂, —CN, —NO₂, —SH, —OH, —NH₂, —CONH₂, —SO₃H, —SO₂H, or —S(═O)H;

The term “cation derived from an onium group not containing nitrogen” asused herein denotes for example, but without limiting, phosphonium[—P⁺(R′)₃], arsonium [—As⁺(R′)₃], oxonium [—O⁺(R′)₂], sulfonium[—S⁺(R′)₂], selenonium [—Se⁺(R′)₂], telluronium [—Te⁺(R′)₂], stibonium[—Sb⁺(R′)₃], or bismuthonium [—Bi⁺(R′)₃], wherein R′ each independentlyis H, (C₁-C₆)alkyl, phenyl, benzyl, or heterocyclyl. In particularembodiments, the cation derived from an onium group not containingnitrogen is a phosphonium as defined hereinabove.

In certain embodiments, the compound of the present invention is acompound of the formula I, wherein R₁ is —CR₆R₇—, wherein R₆ and R₇ eachindependently is H, halogen, —COH, —COOH, —OCOOH, —OCONH₂, —CN, —NO₂,—SH, —OH, —NH₂, —CONH₂, —SO₃H, —SO₂H, —S(═O)H, or (C₁-C₈)alkyloptionally substituted by one or more groups each independently selectedfrom —COH, —COOH, —OCOOH, —OCONH₂, —CN, —NO₂, —SH, —OH, —NH₂, —CONH₂,—SO₃H, —SO₂H, —S(═O)H, —N⁺(R′)₃ or —P⁺(R′)₃, wherein R′ eachindependently is H, (C₁-C₄)alkyl, phenyl, or benzyl. Particular suchcompounds are those wherein R₆ and R₇ each independently is(C₁-C₈)alkyl, preferably (C₁-C₄)alkyl, or H. In preferred embodiments,R₁ is —CH₂—, i.e., R₆ and R₇ are each H.

In certain embodiments, the compound of the present invention is acompound of the formula I, wherein R₂ and R₃ each independently is H, or(C₁-C₄)alkyl, preferably (C₁-C₂)alkyl, optionally substituted by one ormore groups each independently selected from halogen, —COH, —COOH,—OCOOH, —OCONH₂, —CN, —NO₂, —SH, —OH, —NH₂, —CONH₂, —SO₃H, —SO₂H,—S(═O)H, —N⁺(R′)₃ or —P⁺(R′)₃, wherein R′ each independently is H,(C₁-C₄)alkyl, phenyl, or benzyl. Particular such compounds are thosewherein R₂ and R₃ each independently is (C₁-C₄)alkyl, preferably(C₁-C₂)alkyl, or H. In preferred embodiments, R₂ and R₃ are each H.

In certain embodiments, the compound of the present invention is acompound of the formula I, wherein R₄ and R₅ each independently is(C₂-C₁₀)alkylene optionally substituted and further optionallyinterrupted as defined above. In particular such embodiments, R₄ and R₅each independently is (C₂-C₁₀)alkylene, (C₂-C₈)alkylene or(C₂-C₆)alkylene, optionally substituted by one or more groups eachindependently selected from halogen, —COH, —COOH, —OCOOH, —OCONH₂, —CN,—NO₂, —SH, —OH, —NH₂, —CONH₂, —SO₃H, —SO₂H, —S(═O)H, (C₆)aryl,(C₁-C₄)alkylene-(C₆)aryl, heteroaryl, or (C₁-C₄)alkylene-heteroaryl, andfurther optionally interrupted by one or more identical or differentheteroatoms selected from S, O or N, and/or at least one group eachindependently selected from —NH—CO—, —CO—NH—, —N(C₁-C₈alkyl)-,—N(C₆aryl)-, (C₆)arylenediyl, or heteroarylenediyl. More particular suchcompounds are those wherein R₄ and R₅ each independently is(C₂-C₁₀)alkylene, (C₂-C₈)alkylene or (C₂-C₆)alkylene, optionallyinterrupted by one or more identical or different heteroatoms,preferably one or more 0 atoms.

In certain embodiments, the compound of the present invention is acompound of the formula I, wherein R₄ and R₅ are identical.

In certain embodiments, the compound of the present invention is acompound of the formula I, wherein Y each independently is (i) ammonium[—N⁺(R′)₃] or phosphonium [—P⁺(R′)₃], wherein R′ each independently isH, (C₁-C₆)alkyl, phenyl, benzyl, or heterocyclyl; or (ii) imidazolium,optionally substituted by one or more groups each independently selectedfrom halogen, (C₁-C₆)alkyl, —COH, —COOH, —OCOOH, —OCONH₂, —CN, —NO₂,—SH, —OH, —NH₂, —CONH₂, —SO₃H, —SO₂H, or —S(═O)H. Particular suchcompounds are those wherein Y each independently is ammonium [—N⁺(R′)₃]or phosphonium [—P⁺(R′)₃], wherein R′ each independently is H, methyl,ethyl, or propyl; or 1-methyl-imidazolium-3-yl.

In certain embodiments, the compound of the present invention is acompound of the formula I, wherein n is an integer of 5, 6, 7 or 8,preferably 5 or 6.

In certain embodiments, the compound of the present invention is acompound of the formula I, wherein (i) R₁ is —CR₆R₇—, wherein R₆ and R₇each independently is H, halogen, —COH, —COOH, —OCOOH, —OCONH₂, —CN,—NO₂, —SH, —OH, —NH₂, —CONH₂, —SO₃H, —SO₂H, —S(═O)H, or (C₁-C₈)alkyloptionally substituted by one or more groups each independently selectedfrom —COH, —COOH, —OCOOH, —OCONH₂, —CN, —NO₂, —SH, —OH, —NH₂, —CONH₂,—SO₃H, —SO₂H, —S(═O)H, —N⁺(R′)₃ or —P⁺(R′)₃, wherein R′ eachindependently is H, (C₁-C₄)alkyl, phenyl, or benzyl; (ii) R₂ and R₃ eachindependently is H, or (C₁-C₄)alkyl, preferably (C₁-C₂)alkyl, optionallysubstituted by one or more groups each independently selected fromhalogen, —COH, —COOH, —OCOOH, —OCONH₂, —CN, —NO₂, —SH, —OH, —NH₂,—CONH₂, —SO₃H, —SO₂H, —S(═O)H, —N⁺(R′)₃ or —P⁺(R′)₃, wherein R′ eachindependently is H, (C₁-C₄)alkyl, phenyl, or benzyl; (iii) R₄ and R₅each independently is (C₂-C₁₀)alkylene, (C₂-C₈)alkylene or(C₂-C₆)alkylene, optionally substituted by one or more groups eachindependently selected from halogen, —COH, —COOH, —OCOOH, —OCONH₂, —CN,—NO₂, —SH, —OH, —NH₂, —CONH₂, —SO₃H, —SO₂H, —S(═O)H, (C₆)aryl,(C₁-C₄)alkylene-(C₆)aryl, heteroaryl, or (C₁-C₄)alkylene-heteroaryl, andfurther optionally interrupted by one or more identical or differentheteroatoms selected from S, O or N, and/or at least one group eachindependently selected from —NH—CO—, —CO—NH—, —N(C₁-C₈alkyl)-,—N(C₆aryl)-, (C₆)arylenediyl, or heteroarylenediyl; and (iv) n is aninteger of 5, 6, 7 or 8.

In particular such embodiments, the compound of the invention is acompound of the formula I, wherein R₁ is —CH₂—; R₂ and R₃ are H; and R₄and R₅ each independently is (C₂-C₁₀)alkylene, (C₂-C₈)alkylene or(C₂-C₆)alkylene, optionally interrupted by one or more O atoms. Moreparticular such compounds are those wherein Y each independently is (i)ammonium [—N⁺(R′)₃] or phosphonium [—P⁺(R′)₃], wherein R′ eachindependently is H, (C₁-C₆)alkyl, phenyl, benzyl, or heterocyclyl, butpreferably H, methyl, ethyl or propyl; or (ii) imidazolium, optionallysubstituted by one or more groups each independently selected fromhalogen, (C₁-C₆)alkyl, —COH, —COOH, —OCOOH, —OCONH₂, —CN, —NO₂, —SH,—OH, —NH₂, —CONH₂, —SO₃H, —SO₂H, or —S(═O)H, but preferably1-methyl-imidazolium-3-yl. In preferred such embodiments, n is aninteger of 5 or 6.

Specific cationic pillararenes according to the present invention,including those exemplified herein, are the compounds of the formula I,wherein R₁ is —CH₂—; R₂ and R₃ are H; X is a counter anion as definedabove; and: (i) R₄ and R₅ are —(CH₂)₃—, or —(CH₂)₆—; Y is —N⁺(CH₃)₃,—N⁺(C₂H₅)₃, —P⁺(CH₃)₃, or —P⁺(C₂H₅)₃; and n is 5; or (ii) R₄ and R₅ are—(CH₂)₃—; Y is —N⁺(CH₃)₃, —P⁺(CH₃)₃, or 1-methyl-imidazolium-3-yl; and nis 6.

The cationic pillararenes of the invention, also referred to herein as“biofilm inhibitors”, can be prepared by any suitable procedure andtechnology known in the art, e.g., as exemplified herein and depicted indetail in Schemes 1-3.

In another aspect, the present invention provides a compositioncomprising a carrier and a compound of the formula I as defined in anyone of the embodiments above, but excluding the compounds wherein R₁ is—CH₂—; R₂ and R₃ are H; and: (i) n is 5; R₄ and R₈ are —(CH₂)₂—; and Yis 1-methyl-imidazolium-3-yl or —N⁺(CH₃)₃; (ii) n is 5; R₄ and R₅ are—(CH₂)₃—; and Y is —P⁺(C₄H₉)₃; (iii) n is 5; R₄ and R₅ are —(CH₂)₄—; andY is —N⁺(CH₃)₃; (iv) n is 6; R₄ and R₅ are —(CH₂)₂—; and Y is —N⁺(CH₃)₃;(v) n is 6; R₄ and R₅ are —(CH₂)₄—; and Y is —N⁺(CH₃)₃; (vi) n is 6; R₄and R₅ are —(CH₂)₄—; and Y is 1-pyridinium; or (vii) n is 6; R₄ and R₅are —(CH₂)₂—; and Y is 1-methyl-imidazolium-3-yl. Such compositions maybe inter alia pharmaceutical compositions, wherein said carrier is apharmaceutically acceptable carrier, and the counter anion X is apharmaceutically acceptable anion.

In certain embodiments, the composition of the present inventioncomprises a compound selected from those specifically disclosed herein,i.e., a compound of the formula I, wherein R₁ is —CH₂—; R₂ and R₃ are H;X is a counter anion as defined above; and: (i) R₄ and R₅ are —(CH₂)₃—,or —(CH₂)₆—; Y is —N⁺(CH₃)₃, —N⁺(C₂H₅)₃, —P⁺(CH₃)₃, or —P⁺(C₂H₅)₃; and nis 5; or (ii) R₄ and R₅ are —(CH₂)₃—; Y is —N⁺(CH₃)₃, —P⁺(CH₃)₃, or1-methyl-imidazolium-3-yl; and n is 6.

In a further aspect, the present invention relates to a method forinhibiting or disrupting biofilm formation in an aqueous media or on anobject, or for reducing biofilm existing in an aqueous media or attachedto an object, said method comprising contacting said aqueous media orobject with a compound of the formula I as defined in any one of theembodiments above, including the compounds wherein R₁ is —CH₂—; R₂ andR₃ are H; and: (i) n is 5; R₄ and R₅ are —(CH₂)₂—; and Y is1-methyl-imidazolium-3-yl or —N⁺(CH₃)₃; (ii) n is 5; R₄ and R₅ are—(CH₂)₃—; and Y is —P⁺(C₄H₉)₃; (iii) n is 5; R₄ and R₅ are —(CH₂)₄—; andY is —N⁺(CH₃)₃; (iv) n is 6; R₄ and R₅ are —(CH₂)₂—; and Y is —N⁺(CH₃)₃;(v) n is 6; R₄ and R₅ are —(CH₂)₄—; and Y is —N⁺(CH₃)₃; (vi) n is 6; R₄and R₅ are —(CH₂)₄—; and Y is 1-pyridinium; or (vii) n is 6; R₄ and R₅are —(CH₂)₂—; and Y is 1-methyl-imidazolium-3-yl.

In specific embodiments, the method of the present invention comprisescontacting said aqueous media or object with a compound of the formulaI, wherein R₁ is —CH₂—; R₂ and R₃ are H; R₄ and R₅ are identical andeach one is —(CH₂)₂₋₁₀—, i.e., —(CH₂)₂, —(CH₂)₃, —(CH₂)₄, —(CH₂)₅,—(CH₂)₆, —(CH₂)₇, —(CH₂)₈, —(CH₂)₉, or —(CH₂)₁₀; Y is —N⁺(CH₃)₃,—N⁺(C₂H₅)₃, —P⁺(CH₃)₃, —P⁺(C₂H₅)₃, or 1-methyl-imidazolium-3-yl; n is 5or 6; and X is a counter anion, wherein each one of the combinations ofR₁-R₅, Y, n, and X defined herein represents a specific such compound.

In certain embodiments, the method of the present invention is forinhibiting or disrupting biofilm formation in an aqueous media, i.e., amedium having water in it, or for reducing biofilm existing in saidaqueous media, wherein said contacting comprises dissolving a compoundof the formula I, or a composition comprising it, within said aqueousmedia.

In certain embodiments, the method of the present invention is forinhibiting or disrupting biofilm formation on an object, or for reducingbiofilm attached to said object, wherein said contacting comprisescoating said object with a compound of the formula I, or a compositioncomprising it, or immersing said object within a composition comprisingsaid compound, respectively. The object being “treated” according to themethod of the present invention may be, without being limited to, anobject designed for functioning in water, e.g., the hull of a boat, apipe, a filter, a pump, or a heat-exchanger; a medical implant such as astent; a medical device such as a catheter; or a biomedical pad such asan adhesive bandage. In particular such embodiments, the method of thepresent invention comprises coating said object with a compositioncomprising a compound of the formula I, i.e., with an antifoulingcomposition such as antifouling paints and coatings for use inter aliain the food industry and hospitals. Such paints and coatings may beformulated, e.g., as sprays or hydrogels.

In certain embodiments, the method of the present invention as definedin any one of the embodiments above results in increased sensitivity ofsaid aqueous media or object to a bacteriocide, i.e., a substance thatkills bacteria such as a disinfectant (an antimicrobial agent that isapplied to non-living objects to destroy microorganisms that are livingon the objects), antiseptic, or antibiotic, and optionally furthercomprises contacting said aqueous media or object with saidbacteriocide. According to the present invention, the aqueous media orobject “treated” by the method of the invention can be contacted withsaid compound of the formula I and said bacteriocide either at the sametime (i.e., contacted with a combination of said compound of the formulaI and said bacteriocide) or sequentially at any order.

In yet another aspect, the present invention relates to a pharmaceuticalcomposition for inhibiting or disrupting biofilm, e.g., bacterial orfungal biofilm, formation, or reducing biofilm, said compositioncomprising a pharmaceutically acceptable carrier, and a compound of theformula I as defined in any one of the embodiments above, including thecompounds wherein R₁ is —CH₂—, R₂ and R₃ are H; and: (i) n is 5; R₄ andR₅ are —(CH₂)₂—; and Y is 1-methyl-imidazolium-3-yl or —N⁺(CH₃)₃; (ii) nis 5; R₄ and R₅ are —(CH₂)₃—; and Y is —P⁺(C₄H₉)₃; (iii) n is 5; R₄ andR₅ are —(CH₂)₄—; and Y is —N⁺(CH₃)₃; (iv) n is 6; R₄ and R₅ are—(CH₂)₂—; and Y is —N⁺(CH₃)₃; (v) n is 6; R₄ and R₅ are —(CH₂)₄—; and Yis —N⁺(CH₃)₃; (vi) n is 6; R₄ and R₅ are —(CH₂)₄—; and Y is1-pyridinium; or (vii) n is 6; R₄ and R₅ are —(CH₂)₂—; and Y is1-methyl-imidazolium-3-yl, wherein X is a pharmaceutically acceptableanion, i.e., an anion capable of forming a pharmaceutically acceptablesalt of said compound.

Pharmaceutically acceptable anions include, without limiting, chloride,bromide, iodide, acetate, mesylate, esylate, maleate, fumarate,tartrate, bitartrate, sulfate, p-toluenesulfonate, benzenesulfonate,methanesulfonate, ethanedisulfonate (edisylate), ethanesulfonate(esylate), tosylate, benzoate, acetate, phosphate, carbonate,bicarbonate, succinate, and citrate. Multiple anions can be used in asingle preparation if desired.

In specific embodiments, the pharmaceutical composition of the presentinvention comprises a compound of the formula I, wherein R₁ is —CH₂—; R₂and R₃ are H; R₄ and R₈ are identical and each one is —(CH₂)₂₋₁₀—, i.e.,—(CH₂)₂, —(CH₂)₃, —(CH₂)₄, —(CH₂)₅, —(CH₂)₆, —(CH₂)₇, —(CH₂)₈, —(CH₂)₉,or —(CH₂)₁₀; Y is —N⁺(CH₃)₃, —N⁺(C₂H₅)₃, —P⁺(CH₃)₃, —P⁺(C₂H₅)₃, or1-methyl-imidazolium-3-yl; n is 5 or 6; and X is a pharmaceuticallyacceptable anion, wherein each one of the combinations of R₁-R₅, Y, n,and X defined herein represents a specific such compound.

The pharmaceutical compositions provided by the present invention may beprepared by conventional techniques, e.g., as described in Remington:The Science and Practice of Pharmacy, 19^(th) Ed., 1995. Thecompositions can be prepared, e.g., by uniformly and intimately bringingthe active agent, i.e., the compound of the formula I, into associationwith a liquid carrier, a finely divided solid carrier, or both, andthen, if necessary, shaping the product into the desired formulation.The compositions may be in liquid, solid or semisolid form and mayfurther include pharmaceutically acceptable fillers, carriers, diluentsor adjuvants, and other inert ingredients and excipients. In oneembodiment, the pharmaceutical composition of the present invention isformulated as nanoparticles.

The pharmaceutical compositions can be formulated for any suitable routeof administration, e.g., for parenteral administration such asintravenous, intraarterial, intrathecal, intrapleural, intratracheal,intraperitoneal, intramuscular or subcutaneous administration, topicaladministration, oral or enteral administration, or for inhalation. Incertain embodiments, these compositions are formulated for eithertopical administration or for inhalation.

The pharmaceutical composition of the invention may be in the form of asterile injectable aqueous or oleaginous suspension, which may beformulated according to the known art using suitable dispersing, wettingor suspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxic parenterallyacceptable diluent or solvent. Acceptable vehicles and solvents that maybe employed include, without limiting, water, Ringer's solution andisotonic sodium chloride solution.

The term “topical administration” as used herein refers to externalapplication to, e.g., the skin, scalp, mucous membranes, teeth, andhair. Pharmaceutical compositions for topical administration may thus bein the form of an aqueous solution, a gel, a cream, a paste, a lotion, aspray, a suspension, a powder, a dispersion, a salve, an ointment, aserum, an anhydrous stick, oil based sprays, oil-in-water emulsions orwater-in-oil emulsions. In certain particular embodiments, thepharmaceutical composition of the invention is a dental compositionformulated, e.g., as a mouthwash, toothpaste, a composition for rootcanal cleaning and disinfection, or a filling composition with biofilminhibition/prevention properties. In other particular embodiments, thepharmaceutical composition of the invention is formulated for hygienicwash.

Pharmaceutical compositions according to the present invention, whenformulated for inhalation, may be administered utilizing any suitabledevice known in the art, such as metered dose inhalers, liquidnebulizers, dry powder inhalers, sprayers, thermal vaporizers,electrohydrodynamic aerosolizers, and the like.

Pharmaceutical compositions according to the present invention, whenformulated for administration route other than parenteraladministration, may be in a form suitable for oral use, e.g., astablets, troches, lozenges, aqueous, or oily suspensions, dispersiblepowders or granules, emulsions, hard or soft capsules, or syrups orelixirs. Compositions intended for oral use may be prepared according toany method known to the art for the manufacture of pharmaceuticalcompositions and may further comprise one or more agents selected fromsweetening agents, flavoring agents, coloring agents and preservingagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active agent(s) in admixture withnon-toxic pharmaceutically acceptable excipients, which are suitable forthe manufacture of tablets. These excipients may be, e.g., inertdiluents such as calcium carbonate, sodium carbonate, lactose, calciumphosphate, or sodium phosphate; granulating and disintegrating agents,e.g., corn starch or alginic acid; binding agents, e.g., starch, gelatinor acacia; and lubricating agents, e.g., magnesium stearate, stearicacid, or talc. The tablets may be either uncoated or coated utilizingknown techniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate may be employed. They may also becoated using the techniques described in the U.S. Pat. Nos. 4,256,108,4,166,452 and 4,265,874 to form osmotic therapeutic tablets for controlrelease. The pharmaceutical composition of the invention may also be inthe form of oil-in-water emulsion.

The pharmaceutical compositions of the invention may be formulated forcontrolled release of the active agent. Such compositions may beformulated as controlled-release matrix, e.g., as controlled-releasematrix tablets in which the release of a soluble active agent iscontrolled by having the active diffuse through a gel formed after theswelling of a hydrophilic polymer brought into contact with dissolvingliquid (in vitro) or gastro-intestinal fluid (in vivo). Many polymershave been described as capable of forming such gel, e.g., derivatives ofcellulose, in particular the cellulose ethers such as hydroxypropylcellulose, hydroxymethyl cellulose, methylcellulose or methylhydroxypropyl cellulose, and among the different commercial grades ofthese ethers are those showing fairly high viscosity. In otherconfigurations, the compositions comprise the active agent formulatedfor controlled release in microencapsulated dosage form, in which smalldroplets of the active agent are surrounded by a coating or a membraneto form particles in the range of a few micrometers to a fewmillimeters.

Another contemplated formulation is depot systems, based onbiodegradable polymers, wherein as the polymer degrades, the activeagent is slowly released. The most common class of biodegradablepolymers is the hydrolytically labile polyesters prepared from lacticacid, glycolic acid, or combinations of these two molecules. Polymersprepared from these individual monomers include poly (D,L-lactide)(PLA), poly (glycolide) (PGA), and the copolymer poly(D,L-lactide-co-glycolide) (PLG).

The pharmaceutical compositions of the invention can be provided in avariety of dosages, wherein the actual dose administered will depend onthe state of the individual treated, and will be determined as deemedappropriate by the practitioner.

In still another aspect, the present invention relates to a compound ofthe formula I as defined in any one of the embodiments above, includingthe compounds wherein R₁ is —CH₂—; R₂ and R₃ are H; and: (i) n is 5; R₄and R₅ are —(CH₂)₂—; and Y is 1-methyl-imidazolium-3-yl or —N⁺(CH₃)₃;(ii) n is 5; R₄ and R₅ are —(CH₂)₃—; and Y is —P⁺(C₄H₉)₃; (iii) n is 5;R₄ and R₅ are —(CH₂)₄—; and Y is —N⁺(CH₃)₃; (iv) n is 6; R₄ and R₅ are—(CH₂)₂—; and Y is —N⁺(CH₃)₃; (v) n is 6; R₄ and R₅ are —(CH₂)₄—; and Yis —N⁺(CH₃)₃; (vi) n is 6; R₄ and R₈ are —(CH₂)₄—; and Y is1-pyridinium; or (vii) n is 6; R₄ and R₅ are —(CH₂)₂—; and Y is1-methyl-imidazolium-3-yl, for use in inhibiting or disrupting biofilmformation, or reducing biofilm.

In specific embodiments, the compound used for inhibiting or disruptingbiofilm formation, or for reducing biofilm, is a compound of the formulaI, wherein R₁ is —CH₂—; R₂ and R₃ are H; R₄ and R₅ are identical andeach one is —(CH₂)₂₋₁₀—, i.e., —(CH₂)₂, —(CH₂)₃, —(CH₂)₄, —(CH₂)₅,—(CH₂)₆, —(CH₂)₇, —(CH₂)₈, —(CH₂)₉, or —(CH₂)₁₀; Y is —N⁺(CH₃)₃,—N⁺(C₂H₅)₃, —P⁺(CH₃)₃, —P⁺(C₂H₅)₃, or 1-methyl-imidazolium-3-yl; n is 5or 6; and X is a pharmaceutically acceptable anion, wherein each one ofthe combinations of R₁-R₅, Y, n, and X defined herein represents aspecific such compound.

In certain embodiments, the use of the compound of the formula Iaccording to the present invention results in increased sensitivity toantibiotic treatment.

In yet a further aspect, the present invention relates to a method forinhibiting or disrupting biofilm formation, or reducing biofilm, in anindividual in need thereof, comprising administering to said individuala therapeutically effective amount of a compound of the formula I asdefined in any one of the embodiments above, including the compoundswherein R₁ is —CH₂—; R₂ and R₃ are H; and: (i) n is 5; R₄ and R₅ are—(CH₂)₂—; and Y is 1-methyl-imidazolium-3-yl or —N⁺(CH₃)₃; (ii) n is 5;R₄ and R₅ are —(CH₂)₃—; and Y is —P⁺(C₄H₉)₃; (iii) n is 5; R₄ and R₅ are—(CH₂)₄—; and Y is —N⁺(CH₃)₃; (iv) n is 6; R₄ and R₈ are —(CH₂)₂—; and Yis —N⁺(CH₃)₃; (v) n is 6; R₄ and R₅ are —(CH₂)₄—; and Y is —N⁺(CH₃)₃;(vi) n is 6; R₄ and R₅ are —(CH₂)₄—; and Y is 1-pyridinium; or (vii) nis 6; R₄ and R₅ are —(CH₂)₂—; and Y is 1-methyl-imidazolium-3-yl. Incertain embodiments, this method results in increased sensitivity ofsaid individual to an antibiotic treatment, and optionally furthercomprises administering to said individual a therapeutically effectiveamount of said antibiotic.

The invention will now be illustrated by the following non-limitingExamples.

EXAMPLES Experimental Chemical Syntheses

General methods. Starting materials were purchased from Sigma-Aldrich,Alfa Aesar, TCI, Cambridge Isotope Laboratories, and Bio-Lab Ltd.Chemical reactions were monitored by TLC (Merck, silica gel 60 F254) andthe compounds were purified by SiO₂ flash chromatography (MerckKieselgel 60). ¹H and ¹³C NMR spectra were recorded on 400 and 500 MHzBruker Avance NMR spectrometers at 25° C. Chemical shifts (6) are givenin parts per million (ppm) and spin-spin coupling (J) is given in Hz.The chemical shifts are relative to residual HDO signal (at δ 4.80 ppmfor the ¹H NMR) when the solvent is D₂O, to residual CHCl₃ signal (at δ7.26 ppm for the ¹H NMR and 77.2 ppm for the ¹³C NMR) when the solventis CDCl₃, or to residual DMSO (at δ 2.50 ppm for the ¹H NMR and 39.5 ppmfor the ¹³C NMR) when the solvent is DMSO. Determination of C, H, and Ncompositions were performed using the Perkin-Elmer 2400 series IIAnalyzer. High-resolution electrospray mass spectra were recorded on aWaters Synapt instrument.

Compound 5 (Adiri et al. (2013). In the final step, to a solution of 4(50 mg, 42 μmol) in water was added sodium hydroxide (17 mg, 0.42 mmol).The solvent was removed by evaporation to afford a white solid (59 mg,100%). ¹H NMR (D₂O): δ 6.73 (s, ArH, 10H), 4.46 (d, J=16 Hz, ArCH₂Ar,10H), 4.21 (d, J=16 Hz, ArCH₂Ar, 10H), 3.78 (s, ArOCH₂COONa, 10H) ppm.¹³C NMR: δ 178.7, 150.4, 129.7, 115.5, 68.8, 30.2 ppm.

Compound 6a (Yao et al. (2012). Carbon tetrabromide (19.9 g, 60 mmol)was added in small portions to a solution of1,4-bis(2-hydroxyethoxy)benzene (5.0 g, 25 mmol) and triphenylphosphine(15.7 g, 60 mmol) in anhydrous acetonitrile (0.12 L); the reactionmixture was kept at 0° C. during the addition. The resulting mixture wasthen warmed to 25° C. for 4 h under argon atmosphere. The product wasprecipitated by the addition of cold water (0.2 L), and the solid wasfiltered and washed with methanol/water (3:2, 3×100 mL). The product wasrecrystallized from methanol to obtain the title compound as whiteflake-like crystals (5.8 g, 71%). ¹H NMR (400 MHz, CDCl₃): δ 6.86 (s,ArH, 4H), 4.24 (t, J=6.2 Hz, ArOCH₂CH₂Br, 4H), 3.61 (t, J=6.3 Hz,ArOCH₂CH₂Br, 4H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 152.9, 116.1, 68.8,29.4 ppm.

Compound 6b. Carbon tetrachloride (5.8 mL, 60 mmol) was added in smallportions to a solution of 1,4-bis(2-hydroxyethoxy)benzene (5.0 g, 25mmol) and triphenylphosphine (15.7 g, 60 mmol) in anhydrous acetonitrile(0.12 l), and the reaction mixture was kept at 0° C. during theaddition. The resulting mixture was then warmed to 25° C. for 4.5 hoursunder argon atmosphere. The product was precipitated by the addition ofcold water (0.2 l), and the solid was filtered and washed withmethanol/water (3:2, 3×0.1 l). The product was recrystallized frommethanol to obtain 6b as white solid (2.6 g, 44%). ¹H NMR (CDCl₃): δ6.68 (s, ArH, 4H), 4.18 (t, J=5.7 Hz, ArOCH₂CH₂Cl, 4H), 3.78 (t, J=5.7Hz, ArOCH₂CH₂Cl, 4H) ppm. ¹³C NMR: δ 152.9, 116.1, 68.9, 42.1 ppm.

Compound 7a (Yao et al., 2012; Ogoshi et al., 2012). To a solution of 6a(4.0 g, 12 mmol) and paraformaldehyde (1.1 g, 37 mmol) in1,2-dichloroethane (60 mL) was added BF₃·OEt₂ (3.5 g, 25 mmol). Thereaction mixture was kept at 25° C. for 1 h under argon atmosphere. Thereaction mixture was washed with water (2×50 mL) and dried with sodiumsulfate and concentrated in vacuo. The product was purified bychromatography (silica gel; petroleum ether:dichloromethane) to afford7a as a white solid (1.58 g, 38%). ¹H NMR (400 MHz, CDCl₃): δ 6.90 (s,ArH, 10H), 4.22 (t, J=5.7 Hz, ArOCH₂CH₂Br, 20H), 3.84 (s, ArCH₂Ar, 10H),3.62 (t, J=5.7 Hz, ArOCH₂CH₂Br, 20H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ149.8, 129.2, 116.2, 69.1, 30.9, 29.5 ppm.

Compound 7b. To a solution of 6b (2.0 g, 8.5 mmol) and paraformaldehyde(0.76 g, 25 mmol) in 1,2-dichloroethane (30 mL) was added BF₃·OEt₂ (2.4g, 17 mmol). The reaction mixture was kept at 25° C. under argonatmosphere for 1 hour. The reaction mixture was washed with water (2×50mL), brine (2×50 mL) and dried with sodium sulfate. The product waspurified by column chromatography (silica gel; petroleumether:dichloromethane) to afford 7b as white solid (1.1 g, 52%). ¹H NMR(CDCl₃): δ 6.91 (s, ArH, 10H), 4.15 (t, J=5.5 Hz, ArOCH₂CH₂Cl, 20H),3.83 (s, ArCH₂Ar, 10H), 3.80 (t, J=5.5 Hz, ArOCH₂CH₂Cl, 20H) ppm. ¹³CNMR: δ 149.8, 129.1, 115.9, 69.1, 43.2, 29.4 ppm.

Compound 8a (Whiteside et al., 2002). A mixture of hydroquinone (8.0 g,73 mmol), 1,3-dibromopropane (44 g, 0.22 mol), and potassium carbonate(45 g, 0.33 mol) were refluxed in acetone (0.13 L) for 24 hours underargon atmosphere. The reaction mixture was cooled to 25° C. and filteredthrough celite, and the solvent was evaporated under vacuum. The residuewas dissolved in dichloromethane (0.1 L), washed with water (2×50 mL), 3N HCl (2×50 mL), and brine (2×50 mL), dried with sodium sulphate, andconcentrated in vacuo. The product was purified by column chromatography(silica gel; eluent: hexane/ethyl acetate). Further purification byrecrystallization in ethyl acetate/hexane afforded 8a as a white solid(9.5 g, 37%). ¹H NMR (400 MHz, CDCl₃): δ 6.84 (s, ArH, 4H), 4.05 (t,J=5.9 Hz, ArOCH₂CH₂CH₂Br, 4H), 3.60 (t, J=6.7 Hz, ArOCH₂CH₂CH₂Br, 4H),2.29 (m, ArOCH₂CH₂CH₂Br, 4H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 153.0,115.5, 65.9, 32.5, 30.2 ppm.

Compound 9a (Ogoshi et al., 2012). To a solution of 8a (2.5 g, 7.1 mmol)in 1,2-dichloroethane (60 mL) was added paraformaldehyde (0.67 g, 22mmol) followed by BF₃·OEt₂ (1.1 g, 7.8 mmol). The reaction mixture waskept at 30° C. for 30 min under argon atmosphere. The resulting mixturewas cooled to 25° C., and the crude product was precipitated by additionof methanol (0.2 L). The product was purified by chromatography (silicagel; hexane:dichloromethane) to afford the title compound as a whitesolid (1.1 g, 43%). ¹H NMR (400 MHz, CDCl₃): δ 6.74 (s, ArH, 10H), 3.99(t, J=6.3 Hz, ArOCH₂CH₂CH₂Br, 20H), 3.75 (s, ArCH₂Ar, 10H), 3.52 (t,J=6.5 Hz, ArOCH₂CH₂CH₂Br, 20H), 2.21 (m, ArOCH₂CH₂CH₂Br, 20H) ppm. ¹³CNMR (100 MHz, CDCl₃): δ 149.8, 128.5, 115.3, 66.3, 32.7, 30.5, 29.9 ppm.

Compound 10a. To a solution of 6a (2.5 g, 7.7 mmol) and paraformaldehyde(0.46 g, 15 mmol) in chloroform (0.12 mL) was added BF₃·OEt₂ (2.19 g, 15mmol). The reaction mixture was kept at 25° C. under argon atmospherefor 3 hours. The reaction mixture was washed with water (2×100 mL),brine (2×100 mL) and dried with sodium sulfate. The product was purifiedby column chromatography (silica gel; petroleum ether:dichloromethane)to afford 10a as white solid (0.38 g, 15%). ¹H NMR (D₂O): δ 6.78 (s,ArH, 12H), 4.16 (t, J=6.2 Hz, ArOCH₂CH₂Br, 24H), 3.86 (s, ArCH₂Ar, 12H),3.55 (t, J=6.2 Hz, ArOCH₂CH₂Br, 24H) ppm. ¹³C NMR: δ 150.3, 128.6,115.9, 69.1, 30.7, 30.4 ppm.

Compound 11. Trimethylamine (33% in ethanol, 1.0 mL, 5.7 mmol) was addedto a solution of 8a (0.2 g, 0.57 mmol) in ethanol (5 mL). The resultingmixture was refluxed in a pressure tube for 24 hours. After cooling to25° C., the precipitate was filtered, washed with ethanol, and driedunder vacuum to afford white solid (0.26 g, 97%). ¹H NMR (400 MHz, D₂O):δ 6.96 (s, ArH, 4H), 4.11 (t, J=5.8 Hz, ArOCH₂, 4H), 3.52 (m, CH₂CH₂N,4H), 3.12 (s, N(CH₃)₃, 18H), 2.24 (m, ArOCH₂CH₂, 4H) ppm. ¹³C NMR (125MHz, D₂O): δ 153.1, 117.0, 66.2, 64.7, 53.7, 23.4 ppm. HRMS: m/z calcd.for C₁₈H₃₄O₂N₂Br₃ [M+Br]⁻ 547.0170, found 547.0181.

Compound 12. Trimethyl phosphine (1 M in THF, 9.0 mL, 9 mmol) was addedto 8a (0.15 g, 0.43 mmol) under argon atmosphere. The solution wasrefluxed for 24 hours in a pressure tube. The precipitate was filtered,washed with THF, and dried under vacuum to afford a white solid (0.15 g,69%). ¹H NMR (400 MHz, D₂O): 7.00 (s, ArH, 4H), 4.12 (t, J=5.8 Hz,ArOCH₂, 4H), 2.42-2.34 (m, ArOCH₂CH₂, 4H), 2.09-2.04 (m, CH₂P(CH₃)₃,4H), 1.86 (d, J=14.3 Hz, P(CH₃)₃, 18H) ppm. ¹³C NMR (100 MHz, D₂O): δ153.1, 117.0, 68.6 (d, J=16 Hz), 21.7, 21.2 (d, J=54 Hz), 8.0 (d, J=55Hz) ppm. ³¹P NMR (162 MHz, D₂O, H₃PO₄ reference): δ 27.3 ppm. HRMS: m/zcalcd. for C₁₈H₃₄O₂P₂Br₃ [M+Br]⁻ 580.9584, found 580.9599.

Compound 21 (Ma et al., 2011). In the final step, trimethylamine (33% inethanol, 6.4 mL, 24 mmol) was added to a solution of 7a (1.0 g, 0.59mmol) in ethanol (50 mL). The resulting mixture was refluxed for 24hours. After cooling to 25° C., the solvent was removed under vacuum andthe residue was dissolved in water (20 mL). The solution was filteredand the solvent was removed by evaporation to afford colorless solid(1.2 g, 89%). ¹H NMR (D₂O): δ 6.97 (s, ArH, 10H), 4.48 (s, ArOCH₂CH₂N,20H), 3.95 (s, ArCH₂Ar, 10H), 3.83 (s, ArOCH₂CH₂N, 20H), 3.24 (s,N(CH₃)₃, 90H) ppm. ¹³C NMR: δ 150.36, 130.93, 117.49, 65.88, 64.46,55.05, 30.56 ppm.

Compound 22 (Yao et al., 2012; Ogoshi et al., 2012). In the final step,a mixture of 7a (1.2 g, 0.76 mmol) and N-methylimidazole (1.2 g, 15mmol) in toluene was kept at 120° C. for 24 hours. After cooling to 25°C., the solvent was removed under vacuum and the residue wasrecrystallized from ethanol/diethyl ether (1:2) to afford a white solid(1.8 g, 98%). ¹H NMR (D₂O): δ 8.4 (br, Imidazole-H, 10H) 7.63 (s,Imidazole-H, 10H), 7.15 (s, ArH, 10H), 6.76 (s, Imidazole-H, 10H), 4.64(s, Ar—OCH₂CH₂, 20H), 4.47 (s, ArOCH₂CH₂, 20H), 3.66 (s, Imidazole-CH₃,30H), 3.62 (s, ArCH₂Ar, 10H) ppm. ¹³C NMR: δ 150.08, 137.31, 129.94,124.64, 123.46, 116.26, 67.79, 50.25, 36.64, 29.92 ppm.

Compound 23. An excess amount of triethylamine (5.0 mL, 36 mmol) wasadded to a solution of 7a (0.46 g, 0.27 mmol) in ethanol (5.0 mL). Theresulting mixture was refluxed in a pressure tube for 5 days. Aftercooling to 25° C., the product was precipitated by the addition ofdiethyl ether. The precipitate was filtered, and the solid was washedwith diethyl ether and acetone to remove excess triethylamine. The solidwas dissolved in water and concentrated to afford a colorless solid thatturned to thick oil when exposed to air (0.40 g, 54%). ¹H NMR (400 MHz,DMSO-d₆): δ 7.00 (s, ArH, 10H), 4.52 (br, ArOCH₂CH₂N, 20H), 3.98 (br,ArOCH₂CH₂N, 20H), 3.75 (s, ArCH₂Ar, 10H), 3.51 (br, NCH₂CH₃, 60H), 1.26(t, NCH₂CH₃, J=7.1 Hz, 90H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 148.8,128.3, 115.5, 62.1, 55.8, 53.1, 28.7, 7.7 ppm.

Compound 24. 2-Dimethylaminoethanol (0.85 g, 9.5 mmol) was added to asolution of 7a (0.20 g, 0.12 mmol) in ethanol. The resulting mixture wasrefluxed for 48 hours. After cooling to 25° C., the solvent was removedunder vacuum and the residue was dissolved in water (5.0 mL). Thesolution was filtered and the solvent was removed by evaporation.Finally the residue was washed with ethanol and dried under vacuum toafford a light brown solid (0.22 g, 71%). ¹H NMR (D₂O): δ 6.96 (s, ArH,10H), 4.55 (br, ArOCH₂CH₂, 20H), 4.07-3.97 (ArCH₂Ar, ArOCH₂CH₂N &NCH₂CH₂OH, 50H), 3.65 (br, NCH₂CH₂OH, 20H), 3.30 (s, N(CH₃)₂, 60H) ppm.¹³C NMR: δ 150.5, 131.0, 117.6, 67.5, 65.0, 64.5, 56.6, 50.5, 30.8 ppm.

Compound 25. Trimethylamine (33% in ethanol, 0.79 mL, 4.4 mmol) wasadded to a solution of 9a (0.20 g, 0.11 mmol) in ethanol (10 mL). Theresulting mixture was refluxed for 24 hours. After cooling to 25° C.,the solvent was removed under vacuum and the residue was dissolved inwater (5.0 mL). The solution was filtered and the solvent was removed byevaporation. The residue recrystallized as light brown crystals inethanol (0.21 g, 79%). ¹H NMR (D₂O): δ 6.78 (s, ArH, 10H), 3.90 (br,ArOCH₂CH₂CH₂ & ArCH₂Ar, 30H), 3.39 (br, ArOCH₂CH₂CH₂, 20H), 3.10 (br,N(CH₃)₃, 90H), 2.07 (br, ArOCH₂CH₂CH₂, 20H) ppm. ¹³C NMR: δ 150.5,129.9, 116.9, 66.6, 64.4, 53.6, 30.8, 23.4 ppm.

Compound 26. Prepared similarly to compound 21 by reacting 7b (0.15 g,0.12 mmol) with trimethylamine (33% in ethanol, 3.0 mL, 11 mmol) inacetonitrile for 48 h; white solid (0.17 g, 78%). 6 ¹H NMR (400 MHz,D₂O): δ 6.98 (s, ArH, 10H), 4.48 (s, ArOCH₂CH₂N, 20H), 3.95 (s, ArCH₂Ar,10H), 3.84 (s, ArOCH₂CH₂N, 20H), 3.25 (s, N(CH₃)₃, 90H) ppm. ¹³C NMR(100 MHz, D₂O): δ 150.1, 130.7, 117.2, 65.5, 64.1, 54.7, 30.2 ppm.

Compound 27. Prepared similarly to compound 21 by reacting 10a (0.15 g,74 μmol) with trimethylamine (33% in ethanol, 0.70 mL, 3.7 mmol); whitesolid (0.16 g, 73%). ¹H NMR (500 MHz, D₂O): δ 6.94 (s, ArH, 12H), 4.54(t, J=4.9 Hz, ArOCH₂CH₂N, 24H), 3.98 (s, ArCH₂Ar, 12H), 3.78 (t, J=4.8Hz, ArOCH₂CH₂N, 24H), 3.15 (s, N(CH₃)₃, 108H) ppm. ¹³C NMR (125 MHz,D₂O): δ 150.3, 129.6, 116.7, 65.6, 63.9, 54.5, 30.6 ppm.

Compound 28. An excess amount of triethyl amine (5.0 mL, 36 mmol) wasadded to a solution of 9a (0.40 g, 0.22 mmol) in ethanol (5.0 mL). Theresulting mixture was refluxed in a pressure tube for 7 days. Aftercooling to 25° C., the product was precipitated by the addition ofdiethyl ether. The precipitate was filtered, and the solid was washedwith diethyl ether and acetone. The product was sonicated in acetone(5×10 mL) to remove excess triethyl amine. Finally the solid wasdissolved in water and concentrated to afford a white solid (0.42 g,68%). ¹H NMR (400 MHz, DMSO-d₆): δ 6.80 (s, ArH, 10H), 4.19 & 3.89 (br,ArOCH₂CH₂CH₂N, 20H), 3.73 (s, ArCH₂Ar, 10H), 3.44 (br, ArOCH₂CH₂CH₂N,20H), 3.43 (br, NCH₂CH₃, 60H), 2.22 (br, ArOCH₂CH₂CH₂, 20H), 1.26 (t,J=7.0 Hz, NCH₂CH₃, 90H) ppm. ¹³C NMR (100 MHz): δ 148.7, 127.9, 113.7,65.0, 53.6, 52.3, 28.7, 22.4, 7.4 ppm. Anal. calcd. forC₁₂₅H₂₃₀Br₁₀N₁₀O₁₀·10.75H₂O: C, 49.62; H, 8.38; N, 4.63. Found: C,49.31; H, 8.06; N, 4.46.

Compound 29. Trimethylphosphine (1.0 M in THF, 2.2 mL, 2.2 mmol) wasadded to a solution of 9a (0.10 g, 0.05 mmol) in acetonitrile (6.0 mL).The resulting mixture was refluxed in a pressure tube for 96 hours.After cooling to 25° C., the precipitate was filtered, washed withdiethyl ether, and dried under vacuum to afford 29 as white solid (0.12g, 86%). ¹H NMR (400 MHz, DMSO-d₆): δ 6.85 (s, ArH, 10H), 4.07 & 3.90(br, ArOCH₂CH₂CH₂P, 20H), 3.78 (s, ArCH₂Ar, 10H), 2.69-2.60 (m,CH₂P(CH₃)₃, 20H), 2.00 (d, J=14.7 Hz, CH₂P(CH₃)₃& ArOCH₂CH₂CH₂P, 110H)ppm. ¹³C NMR (125 MHz, DMSO-d₆): δ 148.7, 127.8, 114.1, 67.5 (d, J=17Hz), 28.7, 21.7, 19.7 (d, J=56 Hz), 7.5 (d, J=55 Hz) ppm. ³¹p NMR (162MHz, DMSO-d₆, H₃PO₄ reference): δ 30.0 ppm. HRMS: m/z calcd. forC₉₅H₁₇₀O₁₀P₁₀Br₁₁ [M+Br]⁻ 2662.1098, found 2662.1116.

Compound 30. Triethylphosphine (1.0 M in THF, 2.2 mL, 2.2 mmol) wasadded to a solution of 9a (0.10 g, 0.05 mmol) in dry acetonitrile (8.0mL). The resulting mixture was refluxed in a pressure tube for 72 hours.After cooling to 25° C., diethyl ether was added to give a whiteprecipitate. The precipitate was filtered, washed with diethyl ether,and dried under vacuum to afford 30 as white solid (68 mg, 41%). ¹H NMR(500 MHz, DMSO-d₆): δ 6.81 (s, ArH, 10H), 4.15 & 3.84 (br,ArOCH₂CH₂CH₂P, 20H), 3.76 (s, ArCH₂Ar, 10H), 2.61-2.55 (m,CH₂P(CH₂CH₃)₃, 20H), 2.46-2.39 (m, P(CH₂CH₃)₃, 60H), 2.06 (m,OCH₂CH₂CH₂, 20H), 1.23-1.17 (m, P(CH₂CH₃)₃, 90H) ppm. ¹³C NMR (125 MHz,DMSO-d₆): δ 148.7, 127.9, 113.8, 67.5 (d, J=16 Hz), 28.8, 21.7, 14.1 (d,J=50 Hz), 10.8 (d, J=48 Hz), 5.4 (d, J=5 Hz) ppm. ³¹P NMR (162 MHz, D₂O,H₃PO₄ reference): δ 39.8 ppm. Anal. calcd. forC₁₂₅H₂₃₀Br₁₀P₁₀O₁₀·14.45H₂O: C, 45.77; H, 7.74. Found: C, 46.02; H,8.00. HRMS: m/z calcd. for C₁₂₅H₂₃₀O₁₀P₁₀Br₁₁[M+Br]⁻ 3081.5760, found3081.5796.

Biological Assays

Analysis of bacterial growth. The assay was performed as previouslydescribed (Feldman et al., 2012) with minor modifications. Briefly, alltested bacterial strains were grown from the frozen stock in Brain HeartInfusion (BHI) broth for 24 hours at 37° C. in 5% CO₂. Next, 100 μL ofserial 1:2 dilutions of compounds in Tryptic Soy Broth (TSB)+1% glucose(32, 16, 8, 4, 2, 1, and 0.5 μg/mL) were prepared in a flat-bottomed96-well microplates (Costar, Corning). Control wells with no compoundand wells without bacteria containing each tested concentration of thecompounds (blanks) were also prepared. An equal volume (100 μL) ofbacterial suspension diluted 1:100 (OD₆₀₀=0.01) or 1:10 (OD₆₀₀=0.1) inTSB+1% glucose was added to each well. During a 24 hours incubation at37° C., growth kinetics were monitored by recording optical density atwavelength 600 nm (OD₆₀₀) using a Tecan plate reader. Each concentrationwas tested in triplicates, and experiments were repeated three times.

Analysis of biofilm inhibition. The assay was performed as previouslydescribed (Joseph et al., 2016; Feldman et al., 2012) with minormodifications. Briefly, all tested bacterial strains were grown from thefrozen stock in BHI broth for 24 hours at 37° C. in 5% CO₂. Next, 100 μLof serial 1:2 dilutions of compounds in TSB+1% glucose (32, 16, 8, 4, 2,1, and 0.5 μg/mL) were prepared in flat-bottomed 96-well microplates(Costar, Corning). Control wells with no compounds and wells withoutbacteria containing each tested concentration of the compounds (blanks)were also prepared. An equal volume (100 μL) of bacterial suspensionsdiluted 1:100 (OD₆₀₀=0.01) or 1:10 (OD₆₀₀=0.1) in TSB+1% glucose wasadded to each well. After incubation for 24 hours at 37° C. in 5% CO₂under aerobic conditions, spent media and free-floating bacteria wereremoved by turning over the plates. The wells were vigorously rinsed atleast four times with doubly distilled water (DDW). Next, 0.4% crystalviolet (200 μL) was added to each well. After 45 min, wells werevigorously rinsed three times with DDW to remove unbound dye. Afteradding 200 μL of 30% acetic acid to each well, the plate was shaken for15 min to release the dye. Biofilm formation was quantified by measuringthe difference between absorbance of untreated and treated bacterialsamples for each tested concentration of the compounds and theabsorbance of appropriate blank well at 600 nm (A₆₀₀) using Tecan platereader. The MBIC₅₀ was defined as the lowest concentration at which atleast 50% reduction in biofilm formation was measured compared tountreated cells. Each concentration of compound was tested in fivereplicates, and three to six independent experiments were performed.

Analysis of biofilm eradication. Biofilm eradication activity of thetested compounds determined against mature 24-h-old biofilms wasperformed as previously described (Pompilio et al., 2015). Briefly,bacterial species were allowed to form biofilms in TSB+1% glucose mediumin a 96-well flat-bottom microtiter plate by incubation for 24 hours at37° C. in 5% CO₂. Following the 24-h incubation, biofilm samples werewashed twice with sterile DDW, then were exposed to 200 μL of the testedcompounds prepared at concentrations of 2, 4, 8, 16, 32, 64, and 128μg/mL in TSB+1% glucose medium. After incubation at 37° C. in 5% CO₂ for24 h, non-adherent bacteria were removed by washing twice with sterileDDW, and biofilm samples were stained with crystal violet as describedfor biofilm inhibition assay. Untreated biofilm samples were used ascontrol. Biofilms were quantified by measuring the absorbance at 600 nm.The mean IC₅₀ value for biofilm eradication (MBEC₅₀) was defined as thelowest concentration at which at least 50% reduction in biomass ofpreformed biofilms was measured compared to untreated biofilm samples.Concentrations were tested in five replicates, and three independentexperiments were performed.

Rat red blood cell haemolysis assay. A sample of rat red blood cells (2%w/w) were incubated with each of the tested compounds for 1 hours at 37°C. in 5% CO₂ using the double dilution method starting at aconcentration of 256 ug/mL. The negative control was phosphate-bufferedsaline (PBS), and the positive control was 1% w/v solution of TritonX-100 (which induced 100% haemolysis). Following centrifugation (2000rpm, 10 min, ambient temperature), the supernatant was removed andabsorbance at 550 nm was measured using a microplate reader(SpectraMax-M2). The results are expressed as percentage of haemoglobinreleased relative to the positive control (Triton X-100). Experimentswere performed in triplicate, and the results are an average ofexperiments in blood samples taken from at least two rats.

pH stability assay. Aqueous solutions of compounds 28 and 30 weretreated with dilute HCl to obtain a pH of 2.3. Basic pH values of 10.7and 10.2 of solutions of the compounds 28 and 30, respectively, wereobtained by adding dilute ammonia solution. The solutions were kept atroom temperature for 4 hours and then freeze-dried. The ¹H spectrarecorded for all samples matched the ¹H HMR spectra of the respectivecompounds at pH 7.4. Biofilm inhibition studies were conducted withthese compounds as described above.

Mammalian cell toxicity assay. A metabolic activity assay was performedto evaluate toxicity of compounds to human cells in culture aspreviously described (Omata et al., 2006). Briefly, human monocyticTHP-1 cells (ATCC TIB 202) were maintained at 37° C. in 5% CO₂ in RPMI1640 supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100g/mL streptomycin, and 100 units/mL penicillin (all from BiologicalIndustries, Beit HaEmek, Israel). Cystic fibrosis human bronchiepithelial cells IB3-1 (ATCC CRL-2777) and HaCaT human skinkeratinocytes were cultured in Dulbecco's Modified Eagle's Medium (DMEM)medium (Invitrogen) supplemented with 10% FBS, 2 mM glutamine, 100units/mL penicillin, and 100 g/mL streptomycin. Cells were plated in96-well format (60,000 cells/well for THP1; 10,000 cells/well for 1B3-1and HaCaT) for 24 hours at 37° C., 5% CO₂. In Study 1, compound 27 wasadded at final concentrations of 0, 0.73, 2.94, 11.74, and 46.96 μM tothe appropriate wells. In Study 2, compounds were added at finalconcentrations of 2, 4, 8, 16, 32, 64 and 128 μg/ml to the appropriatewells. Wells without compounds served as controls. Plates were incubatedfor 1 hour, 24 hours, and 72 hours at 37° C. in 5% CO₂. Cell viabilitywas determined using MTT (3-[4,5-dimethylthiazoyl-2-yl]-2,5-diphenyltetrazolium bromide) assay (Wataha et al., 1992), and cells wereobserved using light microscopy. The percentage of cell death wasdetermined relative to vehicle-treated cells. Experiments were performedin triplicate, and the results were obtained from two independentexperiments.

Study 1. Cationic Pillararenes Inhibit Formation and Eradicate BacterialBiofilms

In the present study, we prepared five positively charged pillar[5]arenederivatives in which the 10 phenolic positions were substituted bypositively charged quaternary ammonium or imidazolium groups (compounds21-25), and as a control, we prepared the negatively chargedpillar[5]arene 5, which is decorated with 10 carboxylate groups that arenegatively charged under physiological conditions. The compounds weresynthesized following synthetic routes similar to those previouslydescribed (Ma et al., 2011; Yao et al., 2012; Adiri et al., 2013).

Each compound was tested for its ability to inhibit biofilm formation byGram-positive and Gram-negative pathogens. The mean IC₅₀ value forbiofilm inhibition (MBIC₅₀) was defined as the lowest concentration atwhich at least 50% reduction in biofilm formation was measured comparedto untreated cells. The results are summarized in Table 1. The mostimpressive biofilm inhibition properties were observed fordeca-trimethylammonium pillar[5]arene 21 and thedeca-N-methyl-imidazolium pillar[5]arene 22. The MBIC₅₀ values of thesecompounds against each of the tested biofilm-forming Gram-positivepathogens ranged from 0.4 to 6.4 μM. Inhibition of biofilm formation wasselective for Gram-positive strains. None of the cationic pillararenesin this study inhibited the formation of biofilm by Gram-negativestrains E. coli ATCC 25922 and P. aeruginosa PA01.

TABLE 1 Biofilm inhibitory activity of certain pillar[5-6]arenes: MBIC₅₀(μM) against Gram-positive strains* Bacterial strain Compound A B C D EF 21 0.9 3.5 3.5 0.9 3.5 1.8 22 0.8 1.6 1.6 0.4 3.2 6.4 23 1.5 5.9 5.91.5 5.9 5.9 24 >12 >12 >12 >12 >12 >12 25 1.7 6.6 >13 1.7 1.7 6.6 26 1.14.4 8.8 1.1 2.2 8.8 27 0.4 1.5 2.9 0.4 0.7 2.9 5 >23 >23 >23 >23 >23 >23TMA—Cl >292 >292 >292 >292 >292 >292TMA—Br >208 >208 >208 >208 >208 >208 *Compounds were evaluated using thedouble-dilution method for inhibition biofilm formation by (A) S. aureussubsp. aureus Rosenbach ATCC 33592, (B) S. aureus ATCC 29213, (C) S.aureus BAA/043, (D) E. faecalis ATCC 29212, (E) S. epidermidis RP62A,(F) S. mutans ATCC 700610. TMA-Cl and TMA-Br are tetramethylammoniumchloride and tetramethylammonium bromide, respectively. Each value is amean of at least three independent experiments each including fivereplicates of each concentration.

Changes in the hydrophilic or hydrophobic balance of the cationicpillar[5]arene had general and significant effects on the inhibition ofbiofilm formation. Elongation of the aliphatic linker between thepillar[5]arene core and the positively charged group from an ethyl incompound 21 to a propyl chain in compound 25 led to a small butsignificant reduction in the inhibition of biofilm formation. A similareffect was observed when the quaternary tri-methyl ammonium head groupsin compound 21 were replaced by more hydrophobic tri-ethyl quaternaryammonium groups in compound 23. A more pronounced loss of activity wasobserved when the hydrophilicity of the head groups was increased by theinstallation of hydroxyethyl-di-methyl quaternary ammonium groups incompound 24; compound 24 did not inhibit biofilm formation at aconcentration of 12 μM, the highest concentration tested.

To further evaluate the structural determinants required for biofilmformation inhibition, we examined anti-biofilm activities of severalcontrol compounds: Pillar[5]arene 5, which has carboxylic head groupsthat are negatively charged under physiological conditions, did notinhibit biofilm formation by any of the tested strains. This showed thatthe positive charge was important for the observed activity. Noinhibition of biofilm formation, up to concentrations of 292 μM and 208μM, was observed for tetramethylammonium bromide (TMA-Br) ortetramethylammonium chloride (TMA-Cl), respectively, indicating thatneither the quaternary ammonium head groups nor the halogen ions aloneare responsible for the inhibition of biofilm formation by compounds21-25.

The effect of the halogen ion on the inhibition of biofilm formation inthe tested strains was further examined by the preparation of compound26; the chloride analogue of compound 21. Compounds 21 and 26 had verysimilar MBIC50 values against four of the tested strains (strains A, B,D, E; Table 1). The halogen ion type did affect the ability ofpillararene 26 to inhibit S. aureus BAA/043 (strain C) and S. mutansATCC 700610 (strain F) biofilm formation; for these strains, the MBIC50values of compound 26 with the chloride counter ion were two- andfour-fold higher than those of compound 21 with the bromide anion,respectively.

Since 21 demonstrated potent inhibition of biofilm formation by alltested strains of Gram-positive pathogens, we reasoned that increasingthe quaternary ammonium cluster size and the overall positive charge ofthe molecule would further improve the inhibition properties. Hence, wesynthesized compound 27, the pillar[6]arene analogue of 21. Compared tocompound 21 the overall positive charge of compound 27 is 20% higher.Furthermore, the internal cavity diameter of pillar[6]arene 27 is ˜6.7Å, whereas that of pillar[5]arene 21 is ˜4.6 Å (Ogoshi and Yamagishi,2014). This difference should, in principle, enable compound 27 to bindlarger and more structurally diverse molecular guests from the biofilmmatrix. Compound 27 was found to be the most potent inhibitor of biofilmformation of all the cationic pillararenes tested strains as summarizedin Table 1 and demonstrated visually in FIG. 2 . Compared topillar[5]arene 21, the MBIC50 values of pillar[6]arene analogue 27 werefrom 2- to 5-fold lower for four of the tested biofilm formingGram-positive pathogens; no significant difference in the inhibition ofbiofilm formation between 21 and 27 was observed in for S. aureusBAA/043 (strain C, Table 1). For S. mutans ATCC 700610, however,compound 21 was slightly more active than 27 (strain F, Table 1). Thedose-dependent biofilm inhibition ability of compounds 21-27 against E.faecalis ATCC 29212 and S. aureus subsp. aureus Rosenbach ATCC 33592 ispresented in FIG. 3 .

Pillararenes 21 and 27, the most potent inhibitors of biofilm formation,did not eradicate mature biofilms. In addition, we determined the MBIC₅₀of compound 27, which demonstrated potent biofilm inhibition properties,against S. aureus subsp. aureus Rosenbach ATCC 33592, and E. faecalis incultures that were 2-fold, 4-fold, and 10-fold the standard inoculum(OD=0.001). No significant change in MBIC₅₀ values was observedindicating that there is no significant inoculum effect for thiscompound (FIG. 4 ; Table 2).

TABLE 2 Effect of initial inoculum on MBIC₅₀ values of compound 27Bacteria strain Initial inoculum (OD₆₀₀) MBIC₅₀ [μM] S. aureus subsp.0.025 0.37 aureus Rosenbach 0.05 0.37 ATCC 33592 0.1 0.37 E. faecalis0.025 0.37 (ATCC 29212) 0.05 0.73 0.1 0.73

Since all of the pillararenes in this study are cationic amphiphiles, weevaluated the antimicrobial activity of the most potent inhibitor ofbiofilm formation pillar[6]arene 27 to determine whether the capabilityto inhibit biofilm formation results from bactericidal activity. Minimalinhibitory concentration (MIC) experiments were performed following thedouble-dilution protocol (Wiegand et al., 2008; Berkov-Zrihen et al.,2013).

The MIC values against the examined Gram-positive strains were higherthan ˜47 μM, at least 16-fold higher than the highest MBIC50 valuemeasured for pillar[6]arene 27 against the tested strains. We thereforeconcluded that the observed inhibition of biofilm formation did notresult from a bactericidal effect. The possibility that pillar[6]arene27 had a bacteriostatic effect was examined by comparing the growthcurves of two of Gram-negative strains (E. coli ATCC 29522 and P.Aeruginosa PA01) and two Gram-positive strains (S. aureus subsp. aureusRosenbach ATCC 33592 and E. faecalis ATCC 29212) in the absence and inthe presence of 32 and 64 μg/ml of 27 for 24 hours. These concentrationsare ˜15 and ˜30-fold higher than the MBIC50 values measured for thiscompound against the two Gram-positive strains. The growth curvesclearly indicated that, at a concentration significantly higher than theMBIC50, this compound had no effect on bacterial growth (FIG. 5 ). Thus,the anti-Gram-positive biofilm properties of this compound do not resultfrom a bacteriostatic effect.

Finally, many families of antimicrobial cationic amphiphiles disruptmammalian cell membranes as well as bacterial cell membranes(Berkov-Zrihen et al., 2015; Benhamou et al., 2015). Rat red blood cellsserve as a standard model for the evaluation of the ability of compoundsto lyse mammalian cell membranes. Up to a concentration of 94 μM, noneof the cationic pillararenes caused any measurable hemolysis of redblood cells obtained from laboratory rats following a previouslyreported protocol (Benhamou et al., 2015). The toxicity of compound 27toward human monocytic THP1 cells (ATCC TIB 202) and cystic fibrosishuman bronchial epithelial cells IB3-1 (ATCC CRL-2777) was alsoevaluated. No effects on viability were observed after 72-hourincubation with concentrations up to 46.96 μM, about 50 times the MBIC50values measured for this compound against the two Gram-positive strains(FIG. 6 ).

Study 2. Phosphonium Pillar[5]arenes Inhibit Bacterial Biofilm Formation

In the present study, we prepared a series of phosphonium and ammoniumdecorated pillar[5]arenes (25, 28-30) and their respective monomers (11and 12) and studied their anti-biofilm activity with the aim ofevaluating the effect of the nature of the positive charges; thecooperativity of the overall positive charges; and the pillar[n]areneplatform on the observed anti-biofilm activity.

The water-soluble cationic pillar[5]arene derivatives used in this studywere synthesized by a four-step process (Scheme 3). Briefly, in thefirst step commercially available hydroquinone was alkylated with1,3-dibromopropane using potassium carbonate in acetone to afford themonomer 8a. The functionalized pillar[5]arene 9a was obtained by thecyclization of monomer 8a with paraformaldehyde and boron trifluoridediethyletherate in dichloroethane. Reaction of 9a with an excess oftrimethylamine or trimethylamine in ethanol under reflux gave thewater-soluble pillar[5]arene derivatives 25 or 28, respectively, and asimilar procedure was followed to obtain compounds 29 and 30, byreaction with excess of trimethylphosphine or triethylphosphine,respectively (Scheme 4). The control monomers 11 and 12 were synthesizedby reacting 8a with excess trimethylphosphine and trimethylamine,respectively. All the compounds were characterized by ¹H and ¹³C NMR andhigh-resolution mass spectroscopy (HRMS).

The effects of compounds 25 and 28-30 were evaluated on biofilmformation by two clinically important Gram-positive bacterial strains,S. aureus ATCC 33592 and Enterococcus faecalis ATCC 29212. Inhibition ofbiofilm formation was determined using the crystal violet staining assay(Feldman et al., 2012). The minimal concentration at which at least 50%reduction in biofilm formation compared to untreated cells (MBIC₅₀) wasdetermined, and the results are summarized in Table 3. The doseresponses are presented in FIGS. 7-10 .

All the cationic pillar[5]arene derivatives exhibited potent inhibitionof biofilm formation against the two tested Gram-positive pathogens. TheMBIC₅₀ values of the deca-ammonium pillar[5]arene analogues 25 and 28were found to be in the range of 0.71-1.66 μM for both of the testedstrains. The corresponding deca-phosphonium pillar[5]arenes 29 and 30showed a similar range of MBIC₅₀ values, 0.67-1.55 μM. These resultsindicate that replacement of the ammonium cations by phosphonium cationsdoes not significantly affect the inhibition of biofilm formation bycationic pillararenes. Thus, the positive charges are essential for theobserved anti-biofilm activity; however, the nature of the charges has amarginal effect. In this respect, Study 1 shows that a negativelycharged deca-carboxylate derivative of pillar[5]arene does notsignificantly inhibit biofilm formation.

TABLE 3 The anti-biofilm activity (MBIC₅₀) of pillar[5]arene derivatives25 and 28-30* MBIC₅₀ in μM (μg/mL) Compound S. aureus ATCC 33592 E.faecalis ATCC 29212 25 1.66 (4) 1.66 (4) 28 0.71 (2) 1.41 (4) 29 1.55(4) 1.55 (4) 30 1.33 (4) 0.67 (2) 11 >340 (160) >340 (160) 12 >317(160) >317 (160) *Compounds were evaluated using the double-dilutionmethod. Each value is the mean of at least three independent experimentsthat included five replicates at each concentration.

To evaluate the effect of hydrophobicity on the biofilm inhibitionactivity, we compared compounds 28 and 30, in which the ammonium orphosphonium cations are attached to triethyl moieties, to compounds 25and 29, which carry trimethyl moieties. Despite the fact that compounds28 and 30 have 30 more carbon atoms than do compounds 25 and 29, theirMBIC₅₀ values did not significantly differ (Table 3, FIGS. 7A-7B). Inaddition, we found that the dose response for the tested pillar[5]arenederivatives 25 and 28-30 (FIGS. 7A-7B) were also very similar, furthercorroborating the fact that pillararenes 28 and 30 are as effective as25 and 29 in preventing biofilm formation by the two tested strains.

To understand the cumulative effect of the positive charges and theadvantage of clustering these charges on a pillararene scaffold, wesynthesized the monomers 11 and 12, which correspond to the repeatingunits of pillar[5]arenes 25 and 29, respectively. Compounds 11 and 12were also tested for their biofilm inhibition properties towards the twobacterial strains. Monomers 11 and 12 were tested at ˜5-fold higherconcentrations than were compounds 25 and 28-30 such that the numbers ofcharges and ionic strengths of the tested solutions were comparable. Upto 340 μM of 11 and 317 μM of 12 (160 μg/mL of 11 and 12), neither 11nor 12 caused a measurable inhibition of biofilm formation (FIG. 7C),suggesting that in these anti-biofilm agents the cumulative chargeorganization on the pillar[5]arene scaffold is a crucial factor for theobserved activity.

Many cationic amphiphiles act as antimicrobial agents that kill bacteria(Kanazawa et al., 1993 and 1994; Cieniecka-RosIonkiewicz et al., 2005;Kurata et al., 2011; Xue et al., 2015). Therefore, to evaluate whetherthe inhibiting effect of compounds 25 and 28-30 on biofilm formationoriginated from a possible antimicrobial activity of these compounds wemeasured the minimal inhibitory concentrations (MICs) against the testedstrains. The MIC values for compounds 25 and 28-30 were found to be 27,23, 25 and 21 μM, respectively, more than 16 fold higher than thehighest MBIC₅₀ values measured for these compounds against the twotested strains. These results demonstrate that the inhibition of biofilmformation by the phosphonium-decorated pillararenes 29 and 30 did notoriginate from antibacterial activity.

The stability of a bioactive compound may affect the moleculeperformance. To address this issue the stability of the new phosphoniumand ammonium pillar[5]arene derivatives was evaluated by incubation for4 hours in solutions at different pH values. Thereafter the materialswere freeze dried, inspected by ¹H-NMR and tested for their biofilminhibition properties. No significant decomposition was observed in the¹H-NMR spectra recorded after exposure to acidic or alkaline pH (FIG. 11). More importantly, the anti-biofilm activities of compounds 28 and 30remained unchanged after these exposures as seen in Table 4 and FIG. 12.

TABLE 4 Biofilm inhibitory activity against Gram positive strains ofcompounds 20 and 22 after incubation at different pH levels for 4 hoursMBIC₅₀ in μM (μg/mL) S. aureus subsp. aureus E. faecalis Compound pHRosenbach ATCC 33592 ATCC 29212 28 2.3 1.41 (4) 1.41 (4) 7.4 1.41 (4)1.41 (4) 10.7 1.41 (4) 1.41 (4) 30 2.3 0.67 (2) 1.33 (4) 7.4 0.67 (2) 1.33 (4)) 10.2 1.33 (4) 0.67 (2)

Finally, it is well established that many cationic amphiphiles disruptmammalian cell membranes, which limits potential for clinical utility(Jennings et al., 2014). We therefore determined the haemolytic effectof pillar[5]arenes 29 and 30 on rat RBCs. Up to a concentration of 85μM, none of the phosphonium-decorated pillar[5]arenes caused measurablehaemolysis of RBCs. In addition, compounds 25, 29 and 30 were found tohave no effect on mammalian cell viability up to a concentration of 128μg/mL as shown in FIG. 13 .

Study 3. Ammonium Pillar[5-6]arenes Inhibit Bacterial Biofilm Formation

In this study, several ammonium pillar[5-6]arene derivatives other thanthose exemplified in Studies 1-2 were synthesized, utilizing chemicalprocedures similar to those exemplified above, and their biofilminhibition activity was tested as described in the Studies above, usingS. aureus ATCC 33592 (MRSA) and E. faecalis ATCC 29212.

The ammonium pillar[5-6]arene derivatives prepared are compounds 31, 32and 33 and are shown in Scheme 4. Compound 31 is an ammoniumpillar[5]arene derivative similar to compound 25, wherein the spacerrepresented by each one of the groups R₄ and R₅ in the formula I is—(CH₂)₆— rather than —(CH₂)₃— as in compound 25; compound 32 is anammonium pillar[6]arene derivative similar to compound 27, wherein thespacer represented by each one of the groups R₄ and R₅ in the formula Iis —(CH₂)₃— rather than —(CH₂)₂— as in compound 27; and compound 33 isan ammonium pillar[6]arene derivative similar to compound 27, whereinthe cation represented by the group Y in the formula I is1-methyl-imidazolium-3-yl rather than —N⁺(CH₃)₃ as in compound 27.

The biofilm inhibition activity of each one of these compounds is shownin Table 5. Interestingly, the non-symmetric ammonium pillar[5]arenederivatives 34 and 35 (Table 5) were found to have biofilm inhibitionactivity that is remarkably lower than those of the cationicpillar[5-6]arene derivatives of the formula I exemplified in each one ofthe Studies herein, indicating that the presence of cationic groups inboth sides of the compound, and possibly also a symmetric structure, arenecessary for enabling the biofilm inhibition activity.

TABLE 5 The anti-biofilm activity (MBIC₅₀) of pillar[5-6]arenederivatives 31-33 MBIC₅₀ in μM (μg/mL) Compound MRSA E. faecalis 31 0.40.4 32 0.7 0.7 33 0.7 0.7 34 8.6 8.6 35 >15 >15

REFERENCES

-   Adiri, T.; Marciano, D.; Cohen, Y., Chem. Commun., 2013, 49,    7082-7084-   Benhamou, R. I.; Shaul, P.; Herzog, I. M.; Fridman, M., Angew. Chem.    Int. Ed., 2015, 54, 13617-   Berkov-Zrihen, Y.; Herzog, I. M.; Feldman, M.; Sonn-Segev, A.;    Roichman, Y.; Fridman, M., Bioorg. Med. Chem., 2013, 21, 3624-   Berkov-Zrihen, Y.; Herzog, I. M.; Benhamou, R. I.; Feldman, M.;    Steinbuch, K. B.; Shaul, P.; Lerer, S.; Eldar, A.; Fridman, M.,    Chem. Eur. J., 2015, 21, 4340-   Bottcher, T.; Kolodkin-Gal, I.; Kolter, R.; Losick, R.; Clardy,    J., J. Am. Chem. Soc., 2013, 135, 2927-   Chunju, L., Chem. Commun., 2014, 50, 12420-12433-   Cieniecka-RosIonkiewicz, A.; Pernak, J.; Kubis-Feder, J.; Ramani,    A.; Robertson, A. J.; Seddon, K. R., Green Chem. 2005, 7, 855-862-   Costerton, J. W.; Stewart, P. S.; Greenberg, E. P., Science, 1999,    284, 1318-1322-   Cragg, P. J.; Sharma, K., Chem. Soc. Rev., 2012, 41, 597-607-   Davey, M. E.; O'Toole, G. A., Microbiol. Mol. Biol. Rev., 2000, 64,    847-   Davies, D., Nat. Rev. Drug. Discovery, 2003, 2, 114-   Dong, S.; Zheng, B.; Wang, F.; Huang, F., Acc. Chem. Res., 2014a,    47, 1982-1994-   Dong, S.; Yuan, J.; Huang, F., Chem. Sci. 2014b, 5, 247-252-   Feldman, M.; Tanabe, S.; Howell, A.; Garnier, D., BMC Complement.    Altern. Med., 2012, 12, 6-   Fux, C. A.; Costerton, J. W.; Stewart, P. S.; Stoodley, P., Trends    Microbiol., 2005, 13, 34-   Jennings M. C.; Ator, L. E.; Paniak, T. J.; Minbiole K. P. C.;    Wuest, W. M., ChemBioChem, 2014, 15, 2211-2215-   Jie, K.; Yao, Y.; Chi, X.; Huang, F., Chem. Commun. 2014, 50,    5503-5505-   Joseph, R.; Naugolny, A.; Feldman, M.; Herzog, I. M.; Fridman, M.;    Cohen, Y., J. Am. Chem. Soc., 2016, 138, 754-757-   Kanazawa, A.; Ikeda, T.; Endo, T., J. Polym. Sci. Part A Polym.    Chem. 1993, 31, 335-343-   Kanazawa, A.; Ikeda, T.; Endo, T., Antimicrob. Agents Chemother.,    1994, 38, 945-952-   Kurata, S.; Hamada, N.; Kanazawa, A.; Endo, T., Dent. Mater. J.    2011, 30, 960-966-   Li, C., Chem. Commun., 2014, 50, 12420-12433-   Liz, D. G.; Manfredi, A. M.; Medeiros, M.; Montecinos, R.;    Gomez-Gonzalez, B.; Garcia-Rio, L.; Nome, F., Chem. Commun., 2016,    52, 3167-3170-   Ma, Y.; Ji, X.; Xiang, F.; Chi, X.; Han, C.; He, J.; Abliz, Z.;    Chen, W.; Huang, F., Chem. Commun., 2011, 47, 12340-   Ma, Y. J.; Cheng, L.; Li, C.; Mullen, K., Chem. Commun., 2016, 52,    6662-6664-   Mao, X.; Liu, T.; Bi, J.; Luo, L.; Tian, D.; Li, H., Chem. Commun.    2016, 52, 4385-4388-   Nierengarten, I.; Nothisen, M.; Sigwalt, D.; Biellmann, T.; Holler,    M.; Remy, J. S.; Nierengarten, J. F., Chem. Eur. J., 2013, 19,    17552-17558-   Ogoshi, T.; Kanai, S.; Fujinami, S.; Yamagishi, T.; Nakamoto, Y., J.    Am. Chem. Soc., 2008, 130, 5022-5023-   Ogoshi, T.; Ueshima, N.; Yamagishi, T.; Toyota, Y.; Matsumi, N.,    Chem. Commun., 2012, 48, 3536-3538-   Ogoshi, T.; Yamagishi, T., Chem. Commun., 2014, 50, 4776-4787-   Ogoshi, T.; Takashima, S.; Yamagishi, T., J. Am. Chem. Soc., 2015,    137, 10962-10964-   Ogoshi, T.; Akutsu, T.; Shimada, Y.; Yamagishi, T., Chem. Commun.,    2016, 52, 6479-6481-   Omata, Y.; Folan, M.; Shaw, M.; Messer, R. L.; Lockwood, P. E.;    Hobbs, D.; Bouillaguet, S.; Sano, H.; Lewis, J. B.; Wataha, J. C.,    Toxicol In Vitro. 2006, 20, 882-890-   Pompilio, A.; Nicola, S. D.; Crocetta, V.; Guarnieri, S.; Savini,    V.; Carretto, E.; Bonaventura, G. D., BMC Microbiol., 2015, 15, 109-   Rabin, N.; Zheng, Y.; Opoku-Temeg, C.; Du, Y.; Bonsu, E.; Sintim, H.    O., Future Med. Chem., 2015, 7, 493-   Shi, B. B.; Jie, K. C.; Zhou, Y. J.; Zhou, J.; Xia, D. Y.; Huang, F.    H., J. Am. Chem. Soc., 2016, 138, 80-83-   Wang, Q.; Cheng, M.; Zhao, Y.; Wu, L.; Jiang, J.; Wang, L.; Pan, Y.,    Chem. Commun. 2015, 51, 3623-3626-   Wataha, J. C.; Craig, R. G.; Hanks, C. T., Dent. Mater., 1992, 8,    65-71-   Whiteside, M. S.; Kurrasch-Orbaugh, D.; Marona-Lewicka, D.;    Nichols, D. E.; Monte, A., Bioorg. Med. Chem., 2002, 10, 3301-3306-   Wiegand, I.; Hilpert, K.; Hancock, R. E. W., Nat. Protoc., 2008, 3,    163-   Xue, M.; Yang, Y.; Chi, X.; Zhang, Z.; Huang, F., Acc. Chem. Res.,    2012, 45, 1294-1308-   Xue, Y.; Xiao, H.; Zhang, Y., Int. J. Mol. Sci., 2015, 16, 3626-3655-   Yang, J.; Yu, G.; Xia, D.; Huang, F., Chem. Commun., 2014, 50,    3993-3995-   Yao, Y.; Xue, M.; Chi, X.; Ma, Y.; He, J.; Abliz, Z.; Huang, F.,    Chem. Commun., 2012, 48, 6505-   Yao, Y.; Chi, X.; Zhou, T.; Huang, F., Chem. Sci., 2014, 5,    2778-2782-   Zhang, H.; Zhao, Y., Chem. Eur. J., 2013, 19, 16862-16879

What is claimed is:
 1. A method for inhibiting or disrupting biofilmformation, or reducing biofilm, in an individual in need thereof,comprising administering to said individual a therapeutic effectiveamount of a compound of formula I:

wherein R₁ is —CR₆R₇—, wherein R₆ and R₇ each independently is H,halogen, —COR₈, —COOR₈, —OCOOR₈, —OCON(R₈)₂, —CN, —NO₂, —SR₈, —OR₈,—N(R₈)₂, —CON(R₈)₂, —SO₂R₈, —SO₃H, —S(═O)R₈, or (C₁-C₈)alkyl optionallysubstituted by one or more groups each independently selected from thegroup consisting of —COR₈, —COOR₈, —OCOOR₈, —OCON(R₈)₂, —CN, —NO₂, —SR₈,—OR₈, —N(R₈)₂, —CON(R₈)₂, —SO₂R₈, —SO₃H, —S(═O)R₈, —N⁺(R′)₃ and—P⁺(R′)₃, wherein R′ each independently is H, (C₁-C₆)alkyl, phenyl,benzyl, or heterocyclyl, or two R's together with the N atom to whichthey are attached form a 3-7 membered saturated ring, optionallycontaining one or more heteroatoms selected from the group consisting ofO, S and N and optionally further substituted at the additional N atom;R₂ and R₃ each independently is H, halogen, or (C₁-C₈)alkyl optionallysubstituted by one or more groups each independently selected from thegroup consisting of halogen, —COR₈, —COOR₈, —OCOOR₈, —OCON(R₈)₂, —CN,—NO₂, —SR₈, —OR₈, —N(R₈)₂, —CON(R₈)₂, —SO₂R₈, —SO₃H, —S(═O)R₈, —N⁺(R′)₃and —P⁺(R′)₃, wherein R′ each independently is H, (C₁-C₆)alkyl, phenyl,benzyl, or heterocyclyl, or two R's together with the N atom to whichthey are attached form a 3-7 membered saturated ring, optionallycontaining one or more heteroatoms selected from the group consisting ofO, S and N and optionally further substituted at the additional N atom;R₄ and R₅ each independently is selected from the group consisting of(C₁-C₁₀)alkylene, (C₂-C₁₀)alkenylene, and (C₂-C₁₀)alkynylene, optionallysubstituted by one or more groups each independently selected from thegroup consisting of halogen, —COR₈, —COOR₈, —OCOOR₈, —OCON(R₈)₂, —CN,—NO₂, —SR₈, —OR₈, —N(R₈)₂, —CON(R₈)₂, —SO₂R₈, —SO₃H, —S(═O)R₈,(C₆-C₁₀)aryl, (C₁-C₄)alkylene-(C₆-C₁₀)aryl, heteroaryl, and(C₁-C₄)alkylene-heteroaryl, and further optionally interrupted by one ormore identical or different heteroatoms selected from the groupconsisting of S, O and N, or by one or more groups each independentlyselected from the group consisting of —NH—CO—, —CO—NH—, —N(C₁-C₈alkyl)-,—N(C₆-C₁₀aryl)-, (C₆-C₁₀)arylenediyl, and heteroarylenediyl; R₈ eachindependently is H or (C₁-C₈)alkyl; Y each independently is (i) a cationderived from a nitrogen-containing group and selected from the groupconsisting of an ammonium [—N⁺(R′)₃], hydrazinium [—N⁺(R′)₂—N(R′)₂],ammoniumoxy [—N⁺(R′)₂→O], iminium [—N⁺(R′)₂═C<], amidinium[—N⁺(R′)₂—C(R′)═NR′], and guanidinium [—N⁺(R′)₂—C(═NR′)—N(R′)₂]; (ii) acation derived from a nitrogen-containing mono- or polycyclicheteroaromatic group and selected from the group consisting ofpyrazolium, imidazolium, oxazolium, thiazolium, pyridinium,pyrimidinium, quinolinium, isoquinolinium, 1,2,4-triazinium,1,3,5-triazinium, and purinium, optionally substituted by one or moregroups each independently selected from the group consisting of halogen,(C₁-C₆)alkyl, —COH, —COOH, —OCOOH, —OCONH₂, —CN, —NO₂, —SH, —OH, —NH₂,—CONH₂, —SO₃H, —SO₂H, and —S(═O)H; or (iii) a cation derived from anonium group not containing nitrogen and selected from the groupconsisting of phosphonium [—P⁺(R′)₃], arsonium [—As⁺(R′)₃], oxonium[—O⁺(R′)₂], sulfonium [—S⁺(R′)₂], selenonium [—Se⁺(R′)₂], telluronium[—Te⁺(R′)₂], stibonium [—Sb⁺(R′)₃], and bismuthonium [—Bi⁺(R′)₃];wherein R′ each independently is H, (C₁-C₆)alkyl, phenyl, benzyl, orheterocyclyl, or two R's in the ammonium, hydrazinium, ammoniumoxy,iminium, amidinium or guanidinium groups, together with the N atom towhich they are attached, form a 3-7 membered saturated ring, optionallycontaining one or more heteroatoms selected from the group consisting of0, S and N and optionally further substituted at the additional N atom;X is a counter anion; and n is an integer of 5-11.
 2. The method ofclaim 1, wherein: (i) R₁ is —CR₆R₇—, wherein R₆ and R₇ eachindependently is H, halogen, —COH, —COOH, —OCOOH, —OCONH₂, —CN, —NO₂,—SH, —OH, —NH₂, —CONH₂, —SO₃H, —SO₂H, —S(═O)H, or (C₁-C₈)alkyloptionally substituted by one or more groups each independently selectedfrom the group consisting of —COH, —COOH, —OCOOH, —OCONH₂, —CN, —NO₂,—SH, —OH, —NH₂, —CONH₂, —SO₃H, —SO₂H, —S(═O)H, —N⁺(R′)₃ or —P⁺(R′)₃,wherein R′ each independently is H, (C₁-C₄)alkyl, phenyl, or benzyl; or(ii) R₂ and R₃ each independently is H, or (C₁-C₄)alkyl, optionallysubstituted by one or more groups each independently selected from thegroup consisting of halogen, —COH, —COOH, —OCOOH, —OCONH₂, —CN, —NO₂,—SH, —OH, —NH₂, —CONH₂, —SO₃H, —SO₂H, —S(═O)H, —N⁺(R′)₃ and —P⁺(R′)₃,wherein R′ each independently is H, (C₁-C₄)alkyl, phenyl, or benzyl; or(iii) R₄ and R₅ each independently is (C₂-C₁₀)alkylene, optionallysubstituted by one or more groups each independently selected from thegroup consisting of halogen, —COH, —COOH, —OCOOH, —OCONH₂, —CN, —NO₂,—SH, —OH, —NH₂, —CONH₂, —SO₃H, —SO₂H, —S(═O)H, (C₆)aryl,(C₁-C₄)alkylene-(C₆)aryl, heteroaryl, and (C₁-C₄)alkylene-heteroaryl,and further optionally interrupted by one or more identical or differentheteroatoms selected from the group consisting of S, O and N, or by oneor more groups each independently selected from the group consisting of—NH—CO—, —CO—NH—, —N(C₁-C₈alkyl)-, —N(C₆aryl)-, (C₆)arylenediyl, andheteroarylenediyl; or (iv) R₄ and R₅ are identical; or (v) Y eachindependently is (i) ammonium [—N⁺(R′)₃] or phosphonium [—P⁺(R′)₃],wherein R′ each independently is H, (C₁-C₆)alkyl, phenyl, benzyl, orheterocyclyl; or (ii) imidazolium, optionally substituted by one or moregroups each independently selected from the group consisting of halogen,(C₁-C₆)alkyl, —COH, —COOH, —OCOOH, —OCONH₂, —CN, —NO₂, —SH, —OH, —NH₂,—CONH₂, —SO₃H, —SO₂H, and —S(═O)H; or (vi) n is 5, 6, 7, or
 8. 3. Themethod of claim 2, wherein: (i) R₁ is —CH₂—; or (ii) R₂ and R₃ are H; or(iii) R₄ and R₅ each independently is (C₂-C₁₀)alkylene, optionallyinterrupted by one or more 0 atoms; or (iv) Y each independently isammonium [—N⁺(R′)₃] or phosphonium [—P⁺(R′)₃], wherein R′ eachindependently is H, methyl, ethyl, or propyl; or1-methyl-imidazolium-3-yl.
 4. The method of claim 1, wherein: R₁ is—CR₆R₇—, wherein R₆ and R₇ each independently is H, halogen, —COH,—COOH, —OCOOH, —OCONH₂, —CN, —NO₂, —SH, —OH, —NH₂, —CONH₂, —SO₃H, —SO₂H,—S(═O)H, or (C₁-C₈)alkyl optionally substituted by one or more groupseach independently selected from the group consisting of —COH, —COOH,—OCOOH, —OCONH₂, —CN, —NO₂, —SH, —OH, —NH₂, —CONH₂, —SO₃H, —SO₂H,—S(═O)H, —N⁺(R′)₃ and —P⁺(R′)₃, wherein R′ each independently is H,(C₁-C₄)alkyl, phenyl, or benzyl; R₂ and R₃ each independently is H, or(C₁-C₄)alkyl, optionally substituted by one or more groups eachindependently selected from the group consisting of halogen, —COH,—COOH, —OCOOH, —OCONH₂, —CN, —NO₂, —SH, —OH, —NH₂, —CONH₂, —SO₃H, —SO₂H,—S(═O)H, —N⁺(R′)₃ and —P⁺(R′)₃, wherein R′ each independently is H,(C₁-C₄)alkyl, phenyl, or benzyl; R₄ and R₅ each independently is(C₂-C₁₀)alkylene, optionally substituted by one or more groups eachindependently selected from the group consisting of halogen, —COH,—COOH, —OCOOH, —OCONH₂, —CN, —NO₂, —SH, —OH, —NH₂, —CONH₂, —SO₃H, —SO₂H,—S(═O)H, (C₆)aryl, (C₁-C₄)alkylene-(C₆)aryl, heteroaryl, and(C₁-C₄)alkylene-heteroaryl, and further optionally interrupted by one ormore identical or different heteroatoms selected from the groupconsisting of S, O and N, or by one or more groups each independentlyselected from the group consisting of —NH—CO—, —CO—NH—, —N(C₁-C₈alkyl)-,—N(C₆aryl)-, (C₆)arylenediyl, and heteroarylenediyl; and n is 5, 6, 7,or
 8. 5. The method of claim 4, wherein R₁ is —CH₂—; R₂ and R₃ are H;and R₄ and R₈ each independently is (C₂-C₁₀)alkylene, optionallyinterrupted by one or more 0 atoms.
 6. The method of claim 5, wherein Yis (i) ammonium [—N⁺(R′)₃] or phosphonium [—P⁺(R′)₃], wherein R′ eachindependently is H, (C₁-C₆)alkyl, phenyl, benzyl, or heterocyclyl; or(ii) imidazolium, optionally substituted by one or more groups eachindependently selected from the group consisting of halogen,(C₁-C₆)alkyl, —COH, —COOH, —OCOOH, —OCONH₂, —CN, —NO₂, —SH, —OH, —NH₂,—CONH₂, —SO₃H, —SO₂H, and —S(═O)H.
 7. The method of claim 6, wherein Yeach independently is ammonium [—N⁺(R′)₃] or phosphonium [—P⁺(R′)₃],wherein R′ each independently is H, methyl, ethyl, or propyl; or1-methyl-imidazolium-3-yl.
 8. The method of claim 7, wherein n is 5 or6.
 9. The method of claim 8, wherein R₁ is —CH₂—; R₂ and R₃ are H; R₄and R₅ are identical and each one is —(CH₂)₂₋₁₀—; Y is —N⁺(CH₃)₃,—N⁺(C₂H₅)₃, —P⁺(CH₃)₃, —P⁺(C₂H₅)₃, or 1-methyl-imidazolium-3-yl; n is 5or 6; and X is a counter anion.
 10. The method of claim 9, wherein R₁ is—CH₂—; R₂ and R₃ are H; R₄ and R₅ are —(CH₂)₂—; Y is —N⁺(CH₃)₃; n is 5;and X is a counter anion.
 11. The method of claim 1, wherein saidcounter anion is Br⁻, Cl⁻, F⁻, I⁻, PF₆ ⁻, BF₄ ⁻, OH⁻, ClO₄ ⁻, HSO₄ ⁻,CF₃COO⁻, CN⁻, alkylCOO⁻, arylCOO⁻, a pharmaceutically acceptable anion,or a combination thereof.
 12. The method of claim 1, wherein said methodresults in increased sensitivity of said individual to an antibiotictreatment, and optionally further comprises administering to saidindividual a therapeutically effective amount of said antibiotic. 13.The method of claim 1, wherein said compound is formulated for topicaladministration or for inhalation.
 14. The method of claim 13, whereinsaid compound is formulated for topical administration, and said methodcomprises administering said compound to the skin, the scalp, a mucousmembrane, the teeth, or the hair of said individual.
 15. The method ofclaim 13, wherein said compound is formulated for inhalation, and saidmethod is for inhibiting or disrupting biofilm formation, or reducingbiofilm, in the lungs of said individual.